bluuebunny/crossref_metadata_2025 huggingface dataset:
Caffeine‐induced calcium oscillations in heavy‐sarcoplasmic‐reticulum vesicles from rabbit skeletal muscle
Authors: WYSKOVSKY Wolfgang
Published: March 2005

Heavy‐sarcoplasmic‐reticulum vesicles from rabbit skeletal muscle show not only caffeine‐induced calcium release in a medium allowing active calcium loading, but also oscillations in calcium concentration under appropriate conditions. The xanthine derivatives 7‐isobutyl‐1‐methylxanthine and theophylline also induce oscillations under the same conditions. Calcium‐releasing substances with other chemical structures such as adenosine nucleotides or calmodulin antagonists do not induce this effect. With the help of specific inhibitors such as ruthenium red, neomycin or magnesium it was demonstrated that the oscillation mechanism involves the ryanodine receptor/calcium channel. When ATP was substituted by GTP or ITP no oscillations occurred after caffeine application. The subsequent application of ATP, but not of adenosine 5′‐[γ‐thio]triphosphate or adenosine 5′‐[β,γ‐methylene]triphosphate activated the oscillating mechanism, showing ATP to be an essential component of the oscillating system. We investigated the influence of the experimental conditions by altering the caffeine and ATP concentrations, calcium load, pH and ionic strength amongst other parameters. Potassium and anion channels are not involved in calcium oscillations of heavy sarcoplasmic reticulum, nor are the oscillations dependent on membrane potential.
The inner core region of Yersinia enterocolitica Ye75R (0:3) lipopolysaccharide
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , SHASHKOV Alexander S. , STROOBANT Vincent , WARTENBERG Klaus , WARTH Christoph , MAYER Hubert
Published: March 2005

The inner‐core region of the lipopolysaccharide of an UDPGalNAc‐4‐epimerase‐deficient mutant of Yersinia enterocolitica 0:3, designated as Ye75R, was investigated using methylation analysis, 1D‐13C‐NMR and 2D‐13C‐NMR and 1H‐NMR, as well as 31P‐NMR, fast‐atom‐bombardment mass spectrometry (FAB MS) and FAB MS/MS in positive and negative modes. The isolated core heptasaccharide (OS) was composed of 2 units d‐glucose, 3 units ld‐heptose and 1 unit each of dd‐heptose and 3‐deoxy‐d‐manno‐octulosonic acid. Methylation analysis indicated that OS was highly branched with terminal location of the two glucoses and the dd‐heptose unit, which was partially (to about 40%) phosphorylated at C7. These combined studies allowed us to formulate the structure of the inner core region as shown in Scheme 1.

The substitution of the 7‐position of the terminally linked dd‐heptose unit by phosphate could be recognized by MS characterization of permethylated dd‐heptose‐7‐phosphate (alditol acetate) and the extent of the substitution by the ratio of the two well separated 1H signals of dd‐heptose in 500‐MHz 1H‐NMR. Negative FAB MS of OS also indicated the presence of smaller amounts of two hexasaccharides, differing from OS in lacking either one terminal unit of d‐glucose or of the terminal dd‐heptose, and additionally of a pentasaccharide lacking two heptosyl units, namely the terminal dd‐heptose and and the subterminal ld‐heptose. The presence of the smaller oligosaccharides in the OS fraction was also recognized by the methylation analysis.
Structure of the O‐linked carbohydrate chains of porcine zona pellucida glycoproteins
Authors: HOKKE Cornelis H. , DAMM Jan B. L. , PENNINKHOF Bea , AITKEN R. John , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

The N‐linked carbohydrate chains of procine zona pellucida glycoproteins were released by digestion with peptide‐N4‐(N‐acetyl‐β‐glucosaminlyl)asparagine amidase F and subsequently separated from the O‐glycoprotein by gel‐permeation chromatography on Bio‐Gel P‐100. The O‐linked carbohydrate chains were relesed from the O‐glycoprotein by alakaline borohydride treatment. Fractitionation of the extremely heterogeneous mixture of O‐linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel‐permeation chromatography on Bio‐Gel P‐4 and P‐6, anion‐exchange FPLC on Mono Q, and high‐pH anion‐exchange chromatography on CarboPac PA‐1. The primary structures of 32 O‐glycans were determined by one‐ and two‐dimensional 1H‐NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Galβ1‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol, Galβ1‐4(6SO4‐)GlcNAc, and α2‐3‐linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n= 0 to > 6 : [Neu5Gc/Acα2‐3]0‐1[Galβ1‐4(6SO4‐)GlcNAcβ1‐3]nGalβ1‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol.

In addition, smaller compounds were identified in which the Galβ1‐3GalNAc‐ol core is substituted by Neu5Gc/Ac α2‐6‐linked to GalNAc‐ol and/or Neu5Gc/Ac α2‐3‐linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6‐O‐sulfated GlcNAc residues differs from that in the above‐mentioned general structure, and a small portion of the oligosaccharides has the GlcNAcβ1‐3GalNAc‐ol core structure. Analysis of the endo‐β‐galactosidase digests of pools of N‐ and O‐glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N‐acetyllactosamine) chains. In the case of the N‐linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O‐linked carbohydrate chains.
Inhibition of glycogen synthesis by epidermal growth factor in hepatocytes
Authors: PEAK Matthew , AGIUS Loranne
Published: March 2005

Epidermal growth factor (EGF) counteracts the stimulation of glycogen synthesis by insulin in hepatocytes, but it is not known whether this is due to inhibition of glycogen synthesis or to inhibition of the insulin‐signalling mechanism. This study investigates the mechanisms by which EGF affects the basal rate and the insulin stimulation of glycogen synthesis. The basal rate of glycogen synthesis is higher at low than at high cell density. EGF inhibits the basal rate of glycogen synthesis at low cell density but not in confluent cultures and abolishes the difference due to density. However, EGF inhibits the stimulation of glycogen synthesis by insulin irrespective of cell density. Increasing glycogen synthesis by increasing the [glucose] does not abolish the difference in rates of glycogen synthesis due to cell density, neither does it induce responsiveness to EGF at high cell density, establishing that responsiveness to EGF is a function of cell density and not of the basal rate and that inhibition of the insulin stimulation also cannot be accounted for by the higher rate of glycogen synthesis. Cytochalasin D and phalloidin, which alter cell morphology through interactions with the microfilament cytoskeleton, mimic the cell‐density‐dependent inhibition of glycogen synthesis by EGF. The inhibition of glycogen synthesis by EGF and cytochalasin D is additive and cytochalasin D potentiates the inhibition of glycogen synthesis by EGF, suggesting involvement of a cytoskeletal mechanism. Exogenous phospholipase C inhibits glycogen synthesis at both low and high cell density and the inhibition at low cell density is not additive with that caused by either EGF or cytochalasin D, suggesting that these agonists inhibit glycogen synthesis through changes in Ca2+ and/or diacylglycerol. The inhibition of glycogen synthesis by EGF in the absence of insulin stimulation is blocked by neomycin, which inhibits Ca2+ release from intracellular stores but not by antagonists of protein kinase C. It was also inhibited by pertussis toxin (50%), suggesting that it may involve GTP‐binding‐protein‐mediated release of Ca2+ from intracellular stores. The inhibition of the stimulation of glycogen synthesis by insulin was not affected by neomycin and was only marginally inhibited by pertussis toxin or guanosine 5′‐O‐[3‐thio]triphosphate (GTP[S]). We infer from these findings that the inhibition by EGF of the basal rate of glycogen synthesis and of the insulin stimulation are mediated by different mechanisms. The latter is pertussis toxin insensitive and independent of cell density, whereas the former is expressed only at low cell density, it is potentiated by cytochalasin D and inhibited by pertussis toxin.
The complete amino acid sequence of R‐phycocyanin‐I α and β subunits from the red alga Porphyridium cruentum
Authors: DUCRET Axel , SIDLER Walter , FRANK Gerhard , ZUBER Herbert
Published: March 2005

We present here the complete primary structure of R‐phycocyanin‐I α and β subunits from the red alga Porphyridium cruentum. The α chain is composed of 162 amino acid residues (18049Da, calculated from sequence, including chromophore) and carries a phycocyanobilin pigment covalently linked to Cys84. The β chain contains 172 amino acids (19344Da, calculated from sequence, including chromophores) and carries a phycocyanobilin pigment covalently linked at Cys82 and a phycoerythrobilin pigment at Cys153. A γ‐N‐methyl asparagine residue was also characterised at position β72 similar to other phycobiliprotein β subunits. R‐phycocyanin‐I from Porphyridium cruentum shares high sequence identity with C‐phycocyanins (69–83%), R‐phycocyanins (66–70%) and in a less extent with phycoerythrocyanins (57–65%) from various sources.

The presented phylogenetic trees are based on a comparison of all phycobiliprotein amino acid sequences known so far and confirm the clear affiliation of the R‐phycocyanins in the phycocyanin family. In spite of their particular phycobilin pattern, they do not represent intermediate forms between the phycocyanin and the phycoerythrin family. Phycoerythrocyanin, a phycocyanin‐related phycobiliprotein adapted to green light harvesting, is also shown to belong to the phycocyanin family. However, the phycoerythrocyanins diverge from phycocyanins in their different function and it is suggested that they should be assigned to a separate group within the phycocyanin family.
Separation of lysosomes and autophagosomes by means of glycyl‐phenylalanine‐naphtylamide, a lysosome‐disrupting cathepsin‐C substrate
Authors: BERG Trond Olav , STRøMHAUG Per Eivind , BERG Trond , SEGLEN Per Ottar
Published: March 2005

In density‐gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LDH). Lysosomes were identified by marker enzymes such as acid phosphatase, or by degradation products from 125I‐tyramine‐cellobiose‐asialoorosomucoid (125I‐TC‐AOM) loaded into the lysosomes by an intravenous injection in vivo 18 h prior to cell isolation. Autophagosomes and lysosomes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated the possibility of using lysosomal enzyme substrates to achieve selective destruction of lysosomes by swelling. Hepatocytes were first incubated for 2 h at 37°C with vinblastine (50 μM) to obtain an accumulation of autophagosomes (to 3–5‐times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl‐phenylalanine‐2‐naphthylamide (GPN), a cathepsin‐C substrate, and methionine‐O‐methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sedimentability of acid phosphatase and a nearly complete loss of 125I‐TC‐AOM sedimentability in substrate‐treated preparations from control or vinblastine‐treated cells. The lysosomes of cells treated with leupeptin or asparagine were largely resistant to the action of GPN, probably as a result of interference with cathepsin‐C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe, as indicated by a reduction in sedimentable LDH, but GPN had no effect on either autophagosomes or mitochondria. The ability of GPN to selectively destroy lysosomes without affecting the autophagosomes of vinblastine‐treated cells should make GPN treatment a useful aid in the purification of rat liver autophagosomes.
Proteins under pressure
Authors: GROSS Michael , JAENICKE Rainer
Published: March 2005

Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (∼11 800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the ‘abyss” is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non‐adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying ‘metabolic dislocation' is unresolved.

Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins and protein conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High‐pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single‐chain proteins undergo pressure‐induced denaturation in the 100‐MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non‐denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self‐organization. Kinetic aspects of its pressure‐induced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure‐induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo‐physiological conditions, with the post‐translational complex as the most pressure‐sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high‐pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.
Isolation, characterization and developmental expression of the ecdysteroid‐induced E75 gene of the wax moth Galleria mellonella
Authors: JINDRA Marek , SEHNAL František , RIDDIFORD Lynn M.
Published: March 2005

Using the cDNA for the Drosophila ecdysteroid‐induced member of the steroid‐hormone‐receptor superfamily, E75 A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75 A cDNA encoding the C‐terminal zinc‐finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA‐binding domain composed of two zinc fingers and designated GmE75 A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA‐binding and ligand‐binding domains of Drosophila E75 A, respectively. The Galleria genomic clone did not encode the N‐terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75 A and GmE75 B, which differed in their N‐termini.

Probes specific for GmE75 A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75 A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75 A expression but not GmE75 B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75 A mRNA was induced by 20‐hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.
Role of hydroxyl radicals in the activation of human platelets
Authors: IULIANO Luigi , PEDERSEN Jens Z. , PRATICÒ Domenico , ROTILIO Giuseppe , VIOLI Francesco
Published: March 2005

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OH°) formed in an extracellular Fenton‐like reaction. H2O2‐induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OH° scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin‐trapped OH° adduct. The role of OH° was confirmed in experiments with exogenously added iron; free or EDTA‐bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OH° depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N‐formyl‐methionyl‐leucyl‐phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as ‘second messengers' during the initial phase of the platelet activation process.
Heterogenous lipid distribution among chlorophyll‐binding proteins of photosystem II in maize mesophyll chloroplasts
Authors: TREMOLIERES Antoine , DAINESE Paola , BASSI Roberto
Published: March 2005

Photosystem II membrane fractions from dark‐adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl β‐D‐maltoside. Chlorophyll‐binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light‐harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll‐protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll‐protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll‐protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra‐centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll‐protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll‐protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll‐protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids.

Concerning the main light‐harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light‐harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26‐kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.
Characterization and molecular cloning of mannose‐binding lectins from the Orchidaceae species Listera ovata, Epipactis helleborine and Cymbidium hybrid
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J.
Published: March 2005

Mannose‐binding lectins were purified from the leaves of three Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad‐leaved helleborine) and Cymbidium hybrid, using affinity chromatography on mannose–Sepharose‐4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12–13 kDa. All of the isolated lectins exhibit exclusive specificity towards mannose.

A cDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for cDNA clones encoding the lectin using colony hybridization. Since N‐terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose‐binding lectin from Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin cDNA clone was used as a probe to screen the L. ovata library. Subsequently, the cDNA clone encoding the L. ovata lectin was used to screen the cDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approximately 50% sequence similarity both at the nucleotide and amino acid level.

The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectins following post‐translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.
Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin
Authors: WONG Kam‐Bo , KE Yi‐Bao , DONG Yi‐Cheng , LI Xiao‐Bing , GUO Yan‐Wen , YEUNG Hin‐Wing , SHAW Pang‐Chui
Published: March 2005

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N‐glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site‐directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition‐state complex. Similar to ricin A, the variant [E160A]trichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back‐up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically. An activity of 1800‐fold less than that of the wild‐type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.
Inhibition and activation studies on sheep liver sorbitol dehydrogenase
Authors: LINDSTAD Rune I. , HERMANSEN Leonila F. , McKINLEY‐McKEE John S.
Published: March 2005

Reversible inhibition and activation, as well as protection against affinity labelling with dl‐2‐bromo‐3‐(5‐imidazolyl)propionic acid, of sheep liver sorbitol dehydrogenase have been studied. The results presented are discussed in terms of enzyme active‐site properties and may have potential applications for drug design.

Kinetics with mainly sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with sorbitol dehydrogenase from other sources as well as with alcohol dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol dehydrogenase, as the catalytic zinc of sorbitol dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit sorbitol dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo‐alcohols and detergents.

Several inhibitors provide competitive protection against enzyme inactivation by dl‐2‐bromo‐3‐(5‐imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme‐inhibitor complexes to be determined. NADH protects noncompetitively against inactivation.

The presence of some binary and ternary enzyme‐NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.
The C‐terminus of lipoprotein lipase is essential for biological function but contains no domain for glycosylphosphatidylinositol anchoring
Authors: BRUIN Taco , GROOT Niels B. , JANSEN Jaap , KASTELEIN John J. P.
Published: March 2005

In this study we present evidence that the C‐terminus of lipoprotein lipase contains no glycosylphosphatidylinositol addition signal and is therefore not a glycosylphosphatidylinositol‐anchored protein. Furthermore, we present additional evidence that the C‐terminus of lipoprotein lipase is essential for biological function.

Flow cytometric analysis and enzyme‐activity monitoring experiments revealed no pool of lipoprotein lipase releasable by phosphatidylinositol‐specific phospholipase present on the membrane of COS cells transfected with the human lipoprotein lipase gene while, in contrast, a heparin‐releasable pool could be demonstrated. [14C]Ethanolamine, a constituent of the glycosylphosphatidylinositol anchor, was not incorporated into lipoprotein lipase during metabolic labeling. C‐terminal deletion mutants were constructed and expressed in COS cells to investigate the presence of glycosylphosphatidylinositol addition signal on the C‐terminus of human lipoprotein lipase (LPL). The specific activities of the mutants M442 [Leu443–Gly448)‐LPL] and M437 [des‐(Cys438–Gly448)‐LPL] were 78% and 59%, respectively, less than the wild type, while the M432 mutant [des‐(Ala433–Gly449)‐LPL] was catalytically inactive. Determination of the stability of the mutants revealed a decreased stability of the M437, compared with wild‐type, whereas M442 showed the same stability. Flow cytometric analysis showed sustained membrane expression for all mutants including the inactive M432 mutant. These results suggest that the C‐terminus of lipoprotein lipase is essential for maintaining intact catalytic activity but is not involved in any posttranslational proteolytic processing, including cleavage of a glycosylphosphatidylinositol addition signal. We therefore conclude that membrane‐binding of the lipase is not mediated by such anchoring.
S‐Thiolation of human endothelial cell glyceraldehyde‐3‐phosphate dehydrogenase after hydrogen peroxide treatment
Authors: SCHUPPE‐KOISTINEN Ina , MOLDÉUS Peter , BERGMAN Tomas , COTGREAVE Ian A.
Published: March 2005

Exposure of human umbilical vein endothelial cells to oxidants such as hydrogen peroxide, tert‐butyl hydroperoxide and diamide has been shown to induce oxidant‐specific S‐thiolation of cellular proteins. In this study one of the main S‐thiolated proteins in hydrogen‐peroxide‐treated cells was identified as the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase. Additionally, we have shown that the post‐translational modification of the cysteinyl thiols of glyceraldehyde‐3‐phosphate dehydrogenase accompanies an inhibition of the enzyme and that both events are simultaneously and rapidly reversed upon the removal of the oxidative stimulus.
UDPgalactose:glycoprotein–N‐acetyl‐d‐galactosamine 3‐β‐d‐galactosyltransferase activity synthesizing O‐glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates
Authors: GRANOVSKY Maria , BIELFELDT Tim , PETERS Stefan , PAULSEN Hans , MELDAL Morten , BROCKHAUSEN Jennifer , BROCKHAUSEN Inka
Published: March 2005

In order to investigate the role of the peptide moiety of glycoproteins in the control of O‐glycan biosynthesis, UDPgalactose:glycoprotein–N‐acetyl‐d‐galactosamine 3‐β‐d‐galactosyltransferase (core 1 β3‐Gal‐T) from rat liver was tested for its specificity towards GalNAc‐containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid‐phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 β3‐Gal‐T compared to benzyl α‐d‐galactosamine as indicated by their relatively high Vmax/Km. The enzyme preferred threonine α‐d‐galactosamine Thr(GalNAc) to serine α‐d‐galactosamine. Pro on the carboxy‐terminal side adjacent to Thr(GalNAc) was inhibitory. Negatively charged amino acids on either side showed a low Km; substrates with negatively charged amino acids on the amino‐terminal side were highly efficient substrates, suggesting charge–charge interactions between enzyme and substrate. Galβ1‐3GalNAcα residues adjacent to Thr(GalNAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di‐galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of O‐glycans is also controlled by the structure and glycosylation of the peptide core of substrates.
Ionization state and pKa of pterin‐analogue ligands bound to dihydrofolate reductase
Authors: JEONG Soon‐Seog , GREADY Jill E.
Published: March 2005

The ionization state and pKa of the inhibitor 6,8‐dimethyl‐N5‐deazapterin bound to the recombinant human dihydrofolate reductase (rhH2folate reductase) complex with NADPH was determined by a spectrofluorimetric method. The excitation spectra for bound ligand as a function of pH from 6.1 to 9.7 indicated it was the same cationic form as for unbound ligand, which is protonated on N3. However, the lower limit for the pKa of the bound form was determined to be 9.1, a value about pH 2.5 higher than that for free ligand, indicating that ligand bound to the enzyme is protonated at neutral pH. The excitation spectra for bound ligand as a function of pH were generated by computer simulation by employing corrections for the pH dependence of the proportion of bound ligand (variable Kd; ligand‐dissociation constant) and taking account of the different pKa values for bound and unbound forms. A plot of Kd values against pH showed a bell‐shaped curve indicating that 6,8‐dimethyl‐N5‐deazapterin bound to rhH2folate reductase · NADPH to form a ternary complex of ionised enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand; the spectrofluorimetric results are consistent with the first alternative.
Biosynthesis of gangliosides containing C18:1 and C20:1 [3‐14C]sphingosine after administrating [1‐14C]palmitic acid and [1‐14C]stearic acid to rat cerebellar granule cells in culture
Authors: CHIGORNO Vanna , VALSECCHI Manuela , SONNINO Sandro
Published: March 2005

The biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1‐14C]palmitic and [1‐14C]stearic acids which are the precursors for sphingosine biosynthesis.

The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1‐14C]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1‐14C]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged.

Ceramide‐containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide‐containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine.

Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. & Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14–20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process {C20:0 sphingosine C20:0 ceramide→C20:1 ceramide} occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine.
Correlations between biological activities and conformational properties for human, salmon, eel, porcine calcitonins and Elcatonin elucidated by CD spectroscopy
Authors: SILIGARDI Giuliano , SAMORÍ Bruno , MELANDRI Sonia , VISCONTI Marco , DRAKE Alex F.
Published: March 2005

Calcitonin (CT) inhibits osteoclastic bone resorption and induces calcium uptake from body fluids. A comparative study of the conformational behaviours of therapeutic calcitonins [salmon (s), eel (e), a synthetic eel calcitonin analogue (Elcatonin), porcine (p) and human (h) calcitonins] as a function of solvent polarity and temperature have been performed by circular dichroism spectroscopy. Elements of secondary structure were lacking in H2O but could be observed in 2,2,2‐trifluoroethanol and sodium dodecyl sulphate. In particular, similar amounts of α‐helical content (four α‐helical turns) were estimated in trifluoroethanol despite the considerable differences in amino acid sequences. The relative ability to form an α helix, assessed by trifluoroethanol/H2O titration, was found to be Elcatonin > sCT > pCT > eCT > hCT. In Elcatonin, sCT, pCT and eCT the four α‐helical turns were promoted almost completely in a single step, between 0 and 35% trifluoroethanol, unlike hCT where helical structure formation has been reported to involve two steps over the whole trifluoroethanol/H2O range [Arvinte, T. & Drake, A. F. (1993) J. Biol. Chem. 268, 6408–6414]. In SDS, which mimics the membrane environment, conformational differences (3–4 helical turns in Elcatonin, sCT, eCT versus one helical turn in pCT, hCT) were observed and correlate well with biological activity (Elcatonin = sCT = eCT > pCT = hCT).

Low‐temperature studies in a cryogenic solvent mixture showed the formation of high α‐helix content (similar to that in trifluoroethanol) in Elcatonin, sCT, eCT and pCT, whilst a left‐handed extended helix (31 helix) was formed in hCT. This is consistent with the hypothesis of ‘linear’ and ‘helical’ calcitonin receptors [Nakanuta, H., Orlowski, R. C. & Epand, R. M. (1990) Endocrinology 127, 163–169].
The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide‐binding sites
Authors: TRAUT Thomas W.
Published: March 2005

From an analysis of current data on 16 protein structures with defined nucleotide‐binding sites consensus motifs were determined for the peptide segments that form such nucleotide‐binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase‐1a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase‐1a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase‐2 contains the conserved aspartate that coordinates the metal ion on Mg‐ATP. Motif Kinase‐3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP‐binding at a separate regulatory site, formed by the motifs Regulatory‐1 and Regulatory‐2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP‐1 and NMP‐2 bind to the pentose and phosphate of the bound ligand. NMP‐1 is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP‐3a is found in kinases for AMP, GMP, and UMP, while NMP‐3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide‐binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the γ‐phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.
Copper‐atom identification in the active and inactive forms of plasma‐derived FVIII and recombinant FVIII‐ΔII
Authors: BIHOREAU Nicolas , PIN Serge , de KERSABIEC Anne‐Marie , VIDOT Françoise , FONTAINE‐AUPART Marie‐Pierre
Published: March 2005

The plasma‐derived factor VIII (pd‐FVIII) circulates as different heterodimers of heavy and light chains associated by a metallic ion still present in the functional activated factor VIII trimer of molecular mass 50000–45000–70000 Da. The chelation of the metal leads to the dissociation of these complexes with a concomitant loss of the procoagulant activity. Until now, this ion has not been directly identified and its role in the structure/function relationships remains unclear. We report the first determination of the nature of this metal using atomic‐absorption spectroscopy with Zeeman effect. A comparative identification was also performed with the new recombinant factor VIII, FVIII‐ΔII. In the different active pd‐FVIII heterodimers (of molecular mass ranging over 210000–80000–90000–80000 Da) and in FVIII‐ΔII, copper was detected. This result is consistent with sequence similarities described between FVIII and copper‐binding proteins. The quantification of the copper content in FVIII‐ΔII and in the corresponding pd‐FVIII dimer of 90000–80000 Da indicated, for both proteins, the presence of one copper ion/mol FVIII. Copper was also identified in the activated FVIII complex and remained in the dimer of 50000–70000 Da generated during FVIII inactivation. Further dissociation into isolated fragments of molecular masses 70000 Da and 50000 Da was concomitant with the loss of the copper ion. No copper was detected in the isolated fragment of molecular mass 45000 Da. These results suggest that the presence of the cation is not directly related to FVIII activity but is an essential structural prerequisite for FVIII heavy‐light‐chain association.
Assembly of functional skeletal muscle troponin complex in Escherichia coli
Authors: MALNIC Bettina , REINACH Fernando C.
Published: March 2005

The production of multi‐subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro. Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E. coli. We demonstrate that the troponin subunits assembled in the cytoplasm of E. coli cell are fully functional. The troponin complex was purified to homogeneity in high yields. When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro. These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules.
No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase
Authors: FIEDLER Franz , HINZ Heide
Published: March 2005

For the hydrolysis of the two glycosidic bonds of fluorescein di‐β‐D‐galactoside (FDG) by β‐galactosidase from Escherichia coli, small [Hofmann, J. & Sernetz, M.(1983) Anal. Biochem. 131, 180–186] to dramatic [Huang, Z. (1991) Biochemistry 30, 8535–8540] deviations from simple stepwise substrate‐intermediate‐product kinetics have been reported. Intermediate channelling, a preferred hydrolysis of the intermediate fluorescein mono‐β‐D‐galactoside (FMG) formed from FDG at the active site and thus in a favourable position for further reaction, has been postulated. As there were reasons to doubt the previous findings and conclusions, the hydrolysis experiments have been repeated at initial FDG concentrations of 7–200 μM, following the concentrations of FDG, FMG and fluorescein with a reliable method, quantitative HPLC, to completion of the reaction. The transient appearance of substantial amounts of the intermediate FMG also in experiments with 200 μM FDG already rules out the existence of the most efficient intermediate channelling deduced by Huang (1991) from measurements of the initially developing fluorescence, incorrectly ascribed to fluorescein. Redetermination of the Michaelis constants for FDG and FMG led to much higher values than those reported previously. Fitting the progress curves by means of nonlinear regression combined with numerical integration of the rate equations resulted in good fits of the normal step‐wise substrate‐intermediate‐product mechanism, without any necessity of assuming a more complex course of the reaction. So one of the rare examples of the hydrolysis of two bonds at a single enzyme‐substrate encounter has been invalidated.
The magnesium–GTP interaction in microtubule assembly
Authors: GROVER Surinder , HAMEL Ernest
Published: March 2005

Microtubule‐associated‐protein‐dependent assembly of tubulin with GDP in the exchangeable site (tubulin‐GDP) can occur with minimal free Mg2+ (<3 μM). This reaction is totally inhibited by EDTA and by GTP concentrations over 2 mM and stimulated by MgCl2. Quantitative aspects of this stimulation are affected by both the Mg2+ and GTP concentrations, but no relationship exists between reaction rates and relative amounts of different magnesium and GTP species. GTP binding to tubulin‐GDP, while maximally stimulated 2–3‐fold by exogenous MgCl2, was inhibited less than 50% by EDTA, and the amount of GTP bound increased as its concentration rose to levels that inhibited polymerization. Studies on the binding of Mg2+ to tubulin‐GDP in the presence and absence of GTP showed that the increase in the amount of tubulin‐associated Mg2+ was substoichiometric to the amount of GTP bound (maximum stoichiometry of additional Mg2+ to GTP bound, 0.7). Upon polymerization the increased Mg2+ content of tubulin was reduced, indicating its loss during GTP hydrolysis. Mg2+ thus plays a critical role in assembly distinct from its enhancement of GTP binding to the exchangeable site. If magnesium is present in trace amounts, this role must either be catalytic during polymerization or limited to nucleation.
Cation binding to a Bacillus (1,3–1,4)‐β‐glucanase Geometry, affinity and effect on protein stability
Authors: KEITEL Thomas , MELDGAARD Morten , HEINEMANN Udo
Published: March 2005

The hybrid Bacillus (1,3–1,4)‐β‐glucanase H(A16‐M), consisting of 16 N‐terminal amino acids derived from the mature form of the B. amyloliquefaciens enzyme and of 198 C‐proximal amino acids from the B. macerans enzyme, binds a calcium ion at a site at its molecular surface remote from the active center [T. Keitel, O. Simon, R. Borriss & U. Heinemann (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291]. X‐ray diffraction analysis at 0.22‐nm resolution of crystals grown in the absence of calcium and in the presence of EDTA shows this site to be occupied by a sodium ion. Whereas the calcium ion has six oxygen atoms in its coordination sphere, two of which are from water molecules, sodium is fivefold coordinated with a fifth ligand belonging to a symmetry‐related protein molecule in the crystal lattice. The affinity of H(A16‐M) for calcium over sodium has been determined calorimetrically. Calcium binding stabilizes the native three‐dimensional structure of the protein as shown by guanidinium chloride unfolding and thermal inactivation experiments. The enhanced enzymic activity of Bacillusβ‐glucanases at elevated temperatures in the presence of calcium ions is attributed to a general stabilizing effect by the cation.
Yolk granules are the major compartment for bullfrog (Rana catesbeiana) oocyte‐specific ribonuclease
Authors: LIAO You‐Di , WANG Jaang‐Jiun
Published: March 2005

Rana catesbeiana ribonuclease (RC‐RNase) is a pyrimidine guanine sequence‐specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, not in other organs. An immunohistochemical assay showed that RC‐RNase was present in the regular yolk granules, but not in forming yolk granules, yolk platelets, pigment granules, mitochondria clouds or the nucleus. The RC‐RNase was restricted to the lateral amorphous area of the yolk granules, and was absent from the central area that has a vitellogenin crystal lattice. The RC‐RNase was extracted from yolk granules by 0.5 M NaCl and purified by dialysis and affinity chromatography. Most of the RC‐RNase (94%) was found in the yolk granules, the rest RC‐RNase (6%) was found in the cytosol in the form of free RNase and latent RNase. The RC‐RNase extracted from yolk granules was further analyzed by immunoprecipitation and RNase activity assay on an SDS/polyacrylamide gel. Our results suggest that the RC‐RNase activity is regulated by both compartmentation and inhibitor binding.