bluuebunny/crossref_metadata_2025 huggingface dataset:
Fuel Choices by Human Platelets in Human Plasma
Authors: Guppy Michael , Abas Lindy , Neylon Cameron , Whisson Maxwell E. , Whitham Susan , Pethick David W. , Niu Xianwa
Published: July 2004

Despite the fact that homogeneous preparations of isolated cells are now being used very effectively to study a range of important biochemical questions, it is still not known what combination of fuels and energy‐producing pathways is used by cells when offered the complex mixture characteristic of plasma or extracellular fluid. We have developed an in vitro system whereby highly purified and functional human platelets are incubated in human plasma that has been minimally modified from its native state. The concentration of platelets and fuels, and the complexity of fuels in the incubation are similar to those in vivo. The preparation thus represents a reasonable approximation of the physiological condition, considering the complex nature of the system being studied. Measurements carried out simultaneously during the incubation are rates of oxygen consumption, lactate production and fuel oxidation. The data allow the calculation of total ATP turnover, and contributions to this turnover by lactate production and the oxidation of individual fuels. Lactate production accounts for 24% of the ATP turnover. The oxidation of glucose and 3‐hydroxybutyrate each account for under 5%, palmitate for 21%, oleate for 7% and acetate for 9%, leaving 32% of the ATP turnover as yet unaccounted for. The results confirm some previous measurements in the literature, but show that data collected under non‐physiological experimental conditions can be misleading.
β‐Xylosidase Activity, Encoded byxlnD, is Essential for Complete Hydrolysis of Xylan byAspergillus Nigerbut not for Induction of the Xylanolytic Enzyme Spectrum
Authors: van Peij Noel N. M. E. , Brinkmann Joep , Vršanská Mária , Visser Jaap , de Graaff Leo H.
Published: July 2004

Two proteins exhibiting β‐d‐xylosidase activity were identified upon fractionation and purification of a culture filtrate of an arabinoxylan‐grownAspergillus niger, A single band of 110 kDa by SDS/PAGE was obtained in both cases and these were active on xylo‐oligosaccharides and on xylan. PartialxlnDcDNA clones were immunochemically identified and isolated from a λ cDNA expression library. Sequence analysis showed that all cDNA clones correspond to a single gene. A genomic clone was isolated and overexpressed inA. nigerandA. nidulans.ThexlnDgene has an ORF of 2412 nucleotides, encodes a protein of 804 amino acids and contains a potential signal peptide of 26 amino acids. This results in a mature protein of 778 amino acids with a predicted molecular mass of 85 kDa and an isoelectric point of 4.5. The protein is N‐glycosylated and contains 15 potential N‐glycosylation sites. Sequence similarity is found with β‐d‐glucosidases both of bacterial and fungal origin. Both β‐xylosidase proteins purified have high activity on the artificial substratep‐nitrophenyl β‐d‐xylopyranoside (XylNp) and a side activity onp‐nitrophenyl α‐l‐arabinofuranoside andp‐nitrophenyl β‐d‐glucopyranoside.A. nigerstrains in which thexlnDgene was disrupted accumulate mainly xylobiose and xylotriose when grown on xylan and have no significant β‐xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular β‐xylosidase.
The Platelet Antigens CD9, CD42 and Integrin αIIbβIIIa Can be Topographically Associated and Transduce Functionally Similar Signals
Authors: Slupsky Joseph R. , Kamigutt Aura S. , Rhodes Nicholas P. , Cawley John C. , Shaw Andrew R. E. , Zuzel Mirko
Published: July 2004

Investigation of the specific effects of different mAb known to stimulate platelets (agonist mAb) is complicated by interaction of the Fc portion of these mAb with the platelet FcγRII. This has led to the conclusion that nearly all agonist‐mAb‐induced activation of platelets is mediated by this receptor. However, the target antigen‐mediated signal can be analysed provided that the effects of FcγRII engagement can either be reduced or eliminated. We have therefore blocked platelet Fcγ RII with IV.3 Fab fragments (an anti‐Fcγ RII mAb), and stimulated the platelets by cross‐linking intact agonist mAb with F(ab′)2 fragments of an Fc‐specific anti‐mouse antibody. By analysing functional platelet responses and protein‐tyrosine phosphorylation, we found that such non‐Fcγ RII‐mediated cross‐linking of CD9, CD42 and glycoprotein (gp) Ilb/IIIa generates closely similar signals. Since this may indicate molecular associations, we analyzed the surface topography of platelets using the chemical cross‐linking agent dithiobis(sulfosuccinimidyl propionate). We found that a proportion of CD9, gpIIb/IIIa and CD42 molecules associate with each other on the platelet surface membrane. Thus, our results suggest that these antigens are able to form a larger molecular complex and induce similar signals. Furthermore, cross‐linking of CD9 and CD42 stimulated thrombasthenic platelets completely lacking gplIb/IIIa. These data therefore indicate that CD9 and CD42 can signal independently of gpIIb/IIIa, and that signals generated by all these molecules may converge on a common pathway.
4‐α‐Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis
Authors: Jeon Beong‐Sam , Taguchi Hayao , Sakai Hiroshi , Ohshima Toshihisa , Wakagi Takayoshi , Matsuzawa Hiroshi
Published: July 2004

4‐α‐Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 900C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase‐resistant and glucoamylase‐sensitive saccharides were produced. On incubation of amylose with the T. litoralis enzyme, glucoamylase‐resistant but a‐amylase‐ sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the T: litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear α‐1,4‐glucan, but also intramolecular transglycosylation to produce cyclic α‐1,4‐glucan (cycloamylose), similarly to potato 4‐α‐glucanotransferase (called disproportionating enzyme). The gene encoding the T. litoralis 4‐α‐glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659‐amino acid protein with a calculated molecular mass of 77 883 Da. The amino acid sequence of the T. litoralis enzyme showed high similarity with those of α‐amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4‐α‐glucanotransferases.
Evidence for the Existence of Multiple Heparan Sulfate Proteoglycans in the Human Glomerular Basement Membrane and Mesangial Matrix
Authors: Groffen Alexander J. A. , Hop Frank W. H. , Trygovason Karl , Dijkman Henri , Assmann Karel J. M. , Veerkamp Jacques H. , Monnens Leo A. H. , Van Den Heuvel Lambert P. W. J.
Published: July 2004

Heparan sulfate proteoglycans (HSPGs) are essential components of the glomerular basement membrane (GBM) carrying a strong anionic charge. A well‐characterized extracellular HSPG is perlecan, ubiquitously expressed in basement membranes. A cDNA construct encoding domains I and II of human perlecan was expressed as a fusion protein with glutathione S‐transferase. This fusion protein was used to generate monoclonal antibody 95J10. We compared the staining pattern of 95J10 with that of M215, a previously prepared mAb that recognizes HSPG isolated from human GBM. In kidney cortex, the anti‐perlecan mAb 95J10 showed a strong staining of the mesangium, Bowman's capsule, the tubular basement membrane, and stained the GBM only slightly. In contrast, M215 predominantly stained the GBM in a linear fashion. Immunoelectron microscopy supported these results, showing concentrations of perlecan in some regions of the GBM, whereas the unidentified M215 antigen was homogenously distributed throughout the GBM. In other human tissues, both antibodies also produced a different staining pattern. Furthermore, a polyclonal antiserum recognizing HSPG isolated from the GBM did not recognize perlecan from EHS tumors. These results provide evidence for the presence of another HSPG in the GBM that is immunologically distinct from perlecan. The absence of perlecan splice variants in the kidney suggests that this component is encoded by a different gene than perlecan. Given its marked expression in the GBM, this component could be a determining factor in the maintenance of selective glomerular permeability.
Functional and Structural Role of the Cytochrome b Subunit of the Membrane‐Bound Hydrogenase Complex of Alcaligenes Eutrophus H16
Authors: Bernhard Michael , Benelli Bruna , Hochkoeppler Alejandro , Zannoni Davide , Friedrich Barbel
Published: July 2004

This study shows that the product of the hoxZ gene of Alcaligenes eutrophus HI6 is a b‐type cytochrome (cytochrome bz, which is essential for anchoring the membrane‐bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H2‐coupled respiration. The hoxZ product is not required for MBH translocation and H2‐dependent reduction of the redox dye, 2,3,5‐triphenyl‐2‐tetrazolium chloride. The lack of cytochrome bz does not affect the electron‐transport activities linked to oxidation of succinate and NADH, although it enhances the electron‐flow rate through the cytochrome‐c oxidase pathway in hoxZδ membranes. We show that the hoxZ product is a dihaem cytochrome b (haems with of Em7.0+ 10 mV and + 166 mV) involved in H2‐dependent electron transfer. We conclude that cytochrome bz, of the A. eutrophus MBH complex is the link necessary for transfer of electrons from H2 to the ubiquinone pool and that it is required for attachment of MBH to the membrane.
A New Class of Biochemical Oscillator Models Based on Competitive Binding
Authors: Ngo Lan G. , Roussel Marc R.
Published: July 2004

It has been noted that single‐enzyme systems can undergo strongly damped transient oscillations. In this paper, we present a nonlinear dynamics analysis of oscillations in undriven chemical systems. This analysis allows us to classify transient oscillations into two groups. In the first group, oscillations arise from rapid oscillatory relaxation to a slower transient relaxation mode. These oscillations are always strongly damped. In the second group, it is the slowest relaxation mode which is implicated in the oscillations so these can be very lightly damped. This second class of oscillations has not previously been studied in enzymology. We show that a remarkably simple single‐enzyme system, namely competitive inhibition with substrate flow, generates transient oscillations which belong to the second class. In an attempt to design an experimentally realizable version of this model, we then discovered a system which is capable of sustained oscillations. In this experimentally realizable model, two substrates compete to bind to a macromolecule. The flow of one substrate is controlled by a simple feedback device. Sustained oscillations are observed over a very wide range of parameters. In both models, oscillations are favored by a wide disparity in rates of binding and dissociation of the two substrates to the macromolecule.
A 54‐kDa Protein Specifically Associates the 3′ Untranslated Region of Three Maternal mRNAs with the Cytoskeleton of the Animal Part of the Paracertrotus Lividus Egg
Authors: Montana Giovanna , Bonura Angela , Romancino Daniele P. , Sbisà Elisabetta , Di Carlo Matta
Published: July 2004

Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis‐and trans‐acting factors are necessary for bep3 mRNA localization, we have looked for in vitro‐specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54‐kDa protein that binds to the 3′UTR of bep3 mRNA. This 54‐kDa protein also permits association of 3′UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54‐kDa protein to 3′UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54‐kDa protein is the only protein able to bind to the 3′UTR of bep3.
Enzymes from Cold‐Adapted Microorganisms — The Class C β‐lactamase from the Antarctic Psychrophile Psychrobacter Immobilis A5
Authors: Feller Georges , Zekhnini Zoubir , Lamotte‐Brasseur Josette , Gerday Charles
Published: July 2004

A heat‐labile β‐lactamase has been purified from culture supernatants of Psychrobacter immobilis A5 grown at 4°C and the corresponding chromosomal ampC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C β‐lactamases. The kinetic parameters of P. immobilisβ‐lactamase for the hydrolysis of some β‐lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three‐dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine‐mediated H‐bonds and aromatic‐aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.
D‐2‐Hydroxy‐4‐Methylvalerate Dehydrogenase from Lactobacillus Delbrueckii Subsp. Bulgaricus— I. Kinetic Mechanism and pH Dependence of Kinetic Parameters, Coenzyme Binding and Substrate Inhibition
Authors: Alvarez Josep A. , Gelpí Josep Li. , Johnsen Keyji , Bernard Nathalie , Delcour Jean , Clarke Anthony R. , Holbrook J. John , Cortés Antonio
Published: July 2004

The steady‐state kinetics of D‐2‐hydroxy‐4‐methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead‐end inhibition techniques. The mechanism is rapid‐equilibrium ordered in the NAD4+ plus D‐2‐hydroxy‐4‐methylvalerate direction, and steady‐state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E‐NADH‐D‐2‐hydroxy‐4‐methylvalerate and E‐NAD+‐2‐oxo‐4‐methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady‐state measurements show that the rate‐limiting step in both directions at pH 8.0 is between formation of the enzyme‐coenzyme‐substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH → NAD+ direction there is a slow, rate‐limiting rearrangement of the E‐NADH‐oxoacid complex while hydride transfer is very fast. The release of NAD+ at pH 8.0 is 200‐times faster than kcat, (NADH → NAD+) whereas the release of NADH is only 5‐times faster than kcat (NAD+→ NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E‐NADH complex) and 6.4 (in the E‐NAD+ complex) which may be the proton donor and acceptor for the chemical reaction.

At pH 6.5 the mechanism changes in the NADH → NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+. In addition the inhibition by excess oxo‐4‐methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D‐2‐hydroxyacids.
Influence of Zn2+ on the Kinetic Events that Contribute to the 500‐kDa Dextran‐Sulfate‐Dependent Activation of Factor XII (Hageman Factor)
Authors: Loiseau Céline , Randriamahazaka Hyacinthe N. , Nigretto Jean‐Maxime
Published: July 2004

The effect of zinc ions on kinetic and equilibrium steps that may contribute to the activation and subsequent autoactivation of human blood coagulation factor XII in the presence of 500‐kDa dextran sulfate was studied. To analyze the results, the expression of the overall autoactivation constant that had been established from the model presented in a previous study was used. Comparison of the kinetics obtained at different levels of zinc, which included amounts lower than the residual concentration introduced by NaCl in the incubation mixture, suggested that the addition of Zn2+ up to 5 μM lowered the mean number of sites available for the binding of factor XII to the surface from 220 to 172 and increased the rate of the first‐order activation of factor XII by one order of magnitude, from (1.6 ± 0.4) × 10−4 s−1 to (8.0 ± 0.4) × 10−4 s−1 in the presence of 550 nM dextran sulfate. Neither the factor XII/surface dissociation constant (1 μM), the apparent catalytic constant, nor the apparent Michaelis‐Menten constant associated with the postulated multi‐stage kinetic sequence were affected by the presence of zinc. Most experimental trends induced by the presence of zinc could be successfully interpreted by using the model, thus reinforcing its reliability under different conditions.
Mode of Primary Binding to Target Membranes and Pore Formation Induced by Vibrio Cholerae Cytolysin (Hemolysin)
Authors: Zitzer Alexander , Palmer Michael , Weller Ulrich , Wassenaar Trudy , Biermann Christine , Tranum‐Jensen Jørgen , Bhakdi Sucharit
Published: July 2004

Vibrio cholerae cytolysin (VCC) is produced by many non‐choleratoxigenic strains of V. cholerae, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro‐toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high‐affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on human erythrocytes, which are less susceptible to the toxin action. At higher concentrations, binding of the toxin occurs to both rabbit and human erythrocytes in a non‐saturable manner. Cell‐bound toxin monomers oligomerize to form supramolecular structures that are seen in the electron microscope as apparently hollow funnels, and oligomerization correlates functionally with the appearance of small transmembrane pores. Osmotic protection experiments indicate that the toxin channels are of finite size with a diameter of 1–2 nm. The mode of action of VCC closely resembles that of classical pore‐forming toxins such as staphylococcal α‐toxin and the aerolysin of Aeromonas hydrophila.
Regulation and Properties of A Fungal Lipase Showing Interfacial Inactivation by Gas Bubbles, or Droplets of Lipid or Fatty Acid
Authors: Stahmann K.‐Peter , Böddecker Theo , Sahm Hermann
Published: July 2004

Ashbya gossypii can grow on triacylglycerol as carbon source. A degradation rate of 0.05 gXg−1 mycelial dry massXh−1 was detected for soybean oil. Although this rate was within the sensitivity range of lipase assays no activity was detectable. On the other hand, extracellular lipase activity could be visualized by clearance halos round the growing mycelium when trioleoylglycerol was emulsified as the sole carbon source in agar plates. Variation of the culture conditions revealed that reduced shaking speed and decreased fat content in the medium led to detectable amounts of lipase in the supernatant of flask cultures. A maximal activity of 800 Ux1−1 was obtained after 32 h of cultivation in flasks containing 1 % yeast extract and incubated at 60 rpm. Because of its pi of 9.0, the enzyme could be purified in a single step by preparative isoelectric focusing. It appeared as a homogeneous protein in analytical isoelectric focusing and SDS/PAGE (Mr, 35000). The lipase was inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures. These effects suggested an interface inactivation. This idea was supported by a stability modulation observed with the surfactant PluronicR F‐68. Inactivation by oleic acid led to an aggregation of the lipase shown by gel filtration. Growth experiments performed under lipase‐stabilizing conditions revealed a negative influence of glucose, glycerol or oleic acid on detectable lipase activity, probably due to a regulation of lipase formation. Inactivation and regulation thus explained the lack of detectable lipase activity in cultures of A. gossypii growing on triacylglycerol.
Acid‐Induced Disassembly of Glutamate Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus Furiosus Occurs Below pH 2.0
Authors: Chiaraluce Roberta , Schwerdtfeger Ruth M. , Scandurra Roberto , Antranikian Garabed , Consalvi Valerio
Published: July 2004

The stability of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus at low pH values has been studied by activity assay, spectroscopic methods, size‐exclusion chromatography and ultracentrifugation analysis. The enzyme is exceptionally stable and at pH 2.0 its hexameric assembly is preserved despite the changes observed in its tertiary structure. Below pH 1.7 dissociation into monomers starts and is accompanied by a progressive loss of tertiary interactions. Dissociation intermediate(s) were not detectable. At pH 2.0 the addition of NaCl causes the same structural changes observed upon further addition of protons. The monomeric state of the enzyme at pH 1.0 shows a significant content of native secondary structure and can be unfolded by guanidinium chloride. The role of electrostatic interactions in the high stability of the enzyme structure at low pH values is discussed.
Cloning and Expression of the mRNA of Human Galectin‐4, an S‐type Lectin Down‐Regulated in Colorectal Cancer
Authors: Rechreche Hocine , Mallo Gustavo V. , Montalto Guiseppe , Dagorn Jean‐Charles , Iovanna Juan L.
Published: July 2004

We are interested in the characterization of genes whose expressions in the colon are modified during colorectal carcinogenesis. Our approach was to establish the phenotype of a colon tumor by partial sequencing of a large number of transcripts, then to select mRNAs of potential interest by differential screening with complex probes from normal or cancerous colon. In this paper, we report the cloning and sequencing of a mRNA strongly underexpressed in colorectal cancer. It corresponded to a protein comprising 323 amino acids, that appeared to be human galectin‐4 on the basis of 76% and 79% amino acid identity to the rat and pig counterparts, respectively. Tissue distribution analysis showed that its expression was restricted to the small intestine, colon and rectum. Galectin‐4 expression was compared in tumor and normal adjacent colon of 19 patients. In 18 patients, the mRNA concentration was 1.5–50‐times lower in the tumor. No significant correlation was observed between decreased expression of galectin‐4 and the degree of differentiation of the tumor or Duke's state. These results suggest that decreased galectin‐4 mRNA expression may be an early event in colon carcinogenesis. Among five cell lines derived from colon carcinoma, only two (HT29 and LS174T) expressed galectin‐4 mRNA.
Bilirubin Oxidation Provoked by Endotoxins Treatment is Suppressed by Feeding Ascorbic Acid in a Rat Mutant Unable to Synthesize Ascorbic Acid
Authors: Yamaguchi Tokio , Hashizume Tsuneo , Tanaka Makiko , Nakayama Mitsuyo , Sugimoto Akiko , Ikeda Saiko , Nakajima Hiroshi , Horio Fumihiko
Published: July 2004

We examined the possibility that bilirubin oxidation is provoked in vivo by using scurvy‐prone ODS‐odlod rats treated with endotoxins (lipopolysaccharide). Recently, bilirubin oxidative metabolites were isolated from human urine and named biotripyrrin‐a and biotripyrrin‐b. In ODS‐odlod rats fed in ascorbicacid‐free diet, the concentration of bilirubin metabolites in urine was increased 7.0‐fold at 3 h after injection of lipopolysaccharide and 4.4‐fold at 10 h compared to the control rats injected with saline. The dietary supplement of ascorbic acid, the physiological antioxidant, suppressed the increase in bilirubin metabolites in urine after lipopolysaccharide injection: concentrations of biotripyrrin‐a and biotripyrrin‐b in urine collected 6.5–10 h after the injection were lower in rats fed an ascorbic‐acid‐supplemented diet than in rats f4ed an ascorbic‐acid‐free diet. Moreover, feeding of ascorbic acid suppressed the hepatic mRNA level of heme oxygenase‐1, the rate‐limiting enzyme of bilirubin biosyntheses, in rats injected with lipopolysaccharide. These findings indicate that bilirubin oxidation is markedly stimulated in lipopolysaccharide‐treated rats and suggest that bilirubin and ascorbic acid have physiologically protective effects against oxidative stress.
A New Kinetic Model for the Mode of Action of Soluble and Membrane‐Immobilized Glutathione Peroxidase from Bovine Erythrocytes — Effects of Selenium
Authors: Carsol Marie Ange , Pouliquen‐Sonaglia Isabella , Lesgards Guy , Marchis‐Mouren Guy , Puigserver Antoine , Santimone Marius
Published: July 2004

Kinetic studies on the oxidative reaction of glutathione by hydrogen peroxide were performed using soluble and membrane‐bound ox erythrocyte glutathione peroxidase of various types. The effects of organic and inorganic selenium on the glutathione peroxidase activity were also examined.

The kinetic behaviour of the enzyme was investigated using a coupled reaction within a relatively large range of hydrogen peroxide and glutathione concentrations. Non‐parallel double‐reciprocal plots were obtained which suggested that a sequential ordered rather than a ping‐pong mechanism was involved. Similar results were obtained with soluble and membrane‐bound enzyme, whatever the type of crosslinking used.

Crosslinking was performed on a nylon support using various alkylating agents and bifunctional molecules. With all three types of immobilized enzyme thus obtained, a slight but significant increase in the Km was observed.

The effects of selenium were then studied. Using soluble enzyme, a slight increase in the activity was observed in the presence of inorganic selenium (sodium selenite) but not with organic selenium (seleno‐l‐methionine).

Inorganic selenium alone was also found to have a slight effect on the membrane‐bound enzyme. An increase in the catalytic efficiency was observed when glutathione peroxidase was bound using lysine as the bifunctional agent and either glutaraldehyde or triethyloxonium tetrafluoroborate as the reticulation agent, after a three‐month period of incubation.
Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria
Authors: García‐Domínguez Mario , Reyes José C. , Florencio Francisco J.
Published: July 2004

The cyanobacterium Synechocystis sp. PCC 6803 contains two genes encoding two different types of glutamine synthetases (GS), glnA and glnN. The first codes for a typical prokaryotic GS type I and the second one codes for a GS type III, different in amino acid sequence to the prokaryotic GSI and the eukaryotic GS11. The glnN gene has been expressed in Escherichia coli and the corresponding protein purified almost to homogeneity (92%). The native enzyme (500 kDa) was composed of six identical subunits with an apparent molecular mass of 80 kDa. The protein was strongly stabilized in the presence of Mn2+ but not with other divalent cations. Biosynthetic activity of GSIII required the same substrates and cofactors as GSI and GSII enzymes. Apparent Km values for ATP, glutamate and ammonium were 0.43 mM, 0.9 mM and 0.19 mM, respectively. The enzyme was weakly inhibited by several amino acids and strongly inhibited by ADP. Synechocystis GSIII was also inhibited by L‐methionine sulfoximine and DL‐phosphinotricin, two transition‐state analogs of the GS reaction mechanism. GSIII has also been purified from nitrogen‐starved Synechocystis 6803 glnA mutant cells, demonstrating that the GS activity, strongly induced under nitrogen starvation in these cells, corresponds to the glnN gene product. In addition, a Synechocystis 6803 glnN mutant lacks the corresponding 80‐kDa protein (GSIII). Polyclonal antibodies specific for GSIII cross‐react with GSIII from other cyanobacteria. In all the strains analysed, levels of GSIII protein increased under nitrogen deficiency. These data suggest that GSIII is specifically required under conditions of nitrogen starvation.
Intracellular Location, Complex Formation, and Function of the Transporter Associated with Antigen Processing in Yeast
Authors: Urlinger Stefanie , Kuchler Karl , Meyer Thomas H. , Uebel Stephan , Tampé Robert
Published: July 2004

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major‐histocompatibility‐complex class‐I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae. Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER. Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a‐factor secretion in a mutant cell line that lacks the yeast mating‐factor transporter Ste6. Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP‐dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47. This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.
Heterologous Expression and Biochemical Characterization of two Functionally Different Type I Fatty Acid Synthases from Brevibacterium Ammoniagenes
Authors: Stuible Hans‐Peter , Meurer Guido , Schweizer Eckhart
Published: July 2004

The coryneform bacterium, Brevibacterium ammoniagenes, contains two structurally related but functionally differentiated type I fatty acid synthases, FAS‐A and FAS‐B. Isolation of homogeneous preparations of both enzymes was achieved by constructing specific fasA and fasB expression systems. In B. ammoniagenes, insertional mutagenesis of fasB allowed the specific production of enzymatically active FAS‐A. The corresponding fasA mutant was not suited for FAS‐B purification as the level of this enzyme was extremely low, in the fasA‐disruptants. Instead, FAS‐B could be efficiently expressed in the heterologous host, Escherichia coli. Using specific antisera against each of the two FAS variants, FAS‐A was shown to be the predominant FAS protein in B. ammoniagenes. In contrast the two enzymes are expressed at comparable rates in E. coli even though the same upstream sequences were associated with fasA and fasB, as in B. ammoniagenes. Due to their differential capacities of being activated to the phosphopantetheine‐containing holo‐enzyme in the heterologous host, only FAS‐B but not FAS‐A exhibited overall FAS activity when isolated from E. coli. Irrespective of their origin, the purified FAS‐A and FAS‐B proteins were indistinguishable with respect to their flavin fluorescence, their subunit size and their sucrose density gradient sedimentation characteristics. Nevertheless, the in vitro products of both enzymes differ characteristically: while FAS‐A synthesizes mainly the 18–carbon fatty acids oleate and stearate with only traces of palmitate, the major product of FAS‐B is palmitic acid. No unsaturated fatty acids are produced by FAS‐B. Thus, the two B ammoniogenes type I fatty acid synthases differ, in spite of their very similar overall protein structure, in both their ability to synthesize oleic acid and in their chain‐length specificities.
Specificity of Serine Proteinase/Serpin Complex Binding to Very‐Low‐Density Lipoprotein Receptor and α2‐Macroglobulin Receptor/Low‐Density‐Lipoprotein‐Receptor‐Related Protein
Authors: Kasza Aneta , Petersen Helle H. , Heegaard Christian W. , Oka Kazuhiro , Christensen Anni , Dubin Adam , Chan Lawrence , Andreasen Peter A.
Published: July 2004

Very‐low‐density lipoprotein receptor (VLDLR) and α2‐macroglobulin receptor/low‐density‐lipoprotein‐receptor‐related protein (α2MR/LRP) are multifunctional endocytosis receptors of the low‐density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type‐1 plasminogen‐activator inhibitor (PAI‐1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and α2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinasetype and tissue‐type plasminogen aqtivators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI‐1, horse leukocyte elastase inhibitor, protein C inhibitor, C1‐inhibitor, α2‐antiplasmin, α1‐proteinase inhibitor, α1‐antichymotrypsin, protease nexin‐1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor‐associated protein (RAP)‐sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS‐1 cells, which have a high endogenous expression of α2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI‐1, protease nexin‐1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/α2‐antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and α2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and α2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.
Denaturation and Reactivation of Dimeric Human Glutathione Reductase
Authors: Nordhoff Axel , Tziatzios Christos , Van Den Broek Jacomina A. , Schott Markus K. , Kalbitzer Hans‐Robert , Becker Katja , Schubert Dieter , Schirmer R. Heiner
Published: July 2004

Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H+→ 2 GSH + NADP+) is an obligatory FAD‐containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunit‐interface mimetics, similarly to well‐studied viral dimeric proteins. However, ab initio folding and/or dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunit‐interface areas, for example synthetic peptide P11, which represents the intersubunit‐contact helix H11. The structure of this peptide, which might assist inhibitor design, was solved by high‐resolution NMR spectroscopy. Residues 440–453, were found to be α helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P11, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular‐dichroism experiments at 223 nm indicated a half‐life of approximately 20 s at 20°C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3–10 μg/ml, cofactor supplementation, temperature (y; 0–32°C), and time (0–72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plain, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 μg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 μg/ml at pH 6.9 and 20°C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436–478) of human GR were found to inhibit refolding with EC50% values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC50% value was 20 μM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm2, appears to be suitable for studying protein folding, dimerization, and prosthetic‐group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.
Modulation of the Catalytic Activity of Brain Succinic Semialdehyde Reductase by Reaction with Pyridoxal 5′‐Phosphate
Authors: Hong Joung Woo , Cho Sung‐Woo , Yoo Jeong‐Suk , Yoo Byung Kwon , Lee Kil Soo , Choi Soo Young
Published: July 2004

An NADPH‐dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5′‐phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5′‐phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5′‐phosphate in the presence and absence of NADPH followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse‐phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal‐5′‐phosphate–binding site as the region containing the sequence I‐L‐E‐N‐I‐Q‐V‐F‐X‐K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5′‐phosphate to a specific lysyl residue at or near the coenzyme‐binding site of the protein.
Identification of POU‐Class Homeobox Genes in a Freshwater Sponge and the Specific Expression of these Genes During Differentiation
Authors: Seimiya Makiko , Watanabe Yoko , Kurosawa Yoshikazu
Published: July 2004

We reported previously the identification of three homeobox‐containing genes, prox1, prox2 and prox3, in sponges [Seimiya, M., Ishiguro, H., Miura, K., Watanabe, Y. & Kurosawa, Y. (1994) Eur. J. Biochem. 221, 219–2251. The transcripts of prox1 and prox2 were identified in cells at all stages of differentiation. In the present study, we have identified two POU‐class homeobox genes, designated spou‐1 and spou‐2, in a freshwater sponge (Ephydatia fluviatilis). These genes each encode a POU‐specific domain and a POU‐type homeodomain. The amino acid sequences of the POU‐specific domain and the POU‐type homeodomain encoded by the spou‐1 gene were 76% and 67% similar to those of the human Pit‐1 gene, respectively. The amino acid sequence of the POU‐specific domain encoded by the spou‐2 gene was also most similar to that encoded by the human Pit‐1 gene among all the POU‐class homeobox genes that have been sequenced to date. In contrast to the results for prox1 and prox2, transcripts of the spou‐1 and spou‐2 genes were identified in cells only at specific stages during the differentiation of the sponge.
Tetrazolium‐Dye‐Linked Alcohol Dehydrogenase of the Methylotrophic Actinomycete Amycolatopsis Methanolica is a Three‐Component Complex
Authors: Bystrykh Leonid V. , Govorukhina Natalya I. , Dijkhuizen Lubbert , Duine Johannis A.
Published: July 2004

Tetrazolium‐dye‐linked alcohol dehydrogenase (TD‐ADH) of Amycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD‐ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N′‐dimethyl‐4‐nitrosoaniline oxidoreductase (MNO), a decameric protein with 50‐kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (>640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazoliumdye‐linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5′‐deazaflavin and at least one other low‐molecular‐mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD‐ADH complex is carried out by component 1 (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5′‐deazaflavin was present in component 3. Other gram‐positive bacteria having a TD‐ADH complex also produced 5′‐deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. methanolica, and probably also by other gram‐positive bacteria containing MNO or TD‐ADH, proceeds via TD‐ADH. The likeliness of 5′‐deazaflavin participation in this process is discussed.
Role of Ribonucleotide Reductase and Deoxynucleotide Pools in the Oxygen‐Dependent Control of DNA Replication in Ehrlich Ascites Cells
Authors: Brischwein Klaus , Engelcke Michael , Riedinger Hans‐Jörg , Probst Hans
Published: July 2004

Cultured Ehrlich ascites cells were exposed to different oxygen tensions (ranging from nearly complete anoxia to 95% O2 at 105 Pa) and to transient (5–10 h) hypoxia (0.02% O2 at 105 Pa). Treated cells were examined with respect to the intracellular concentration of the M2‐specific tyrosyl free radical of ribonucleotide reductase by EPR spectroscopy, and with respect to the pool sizes of all four deoxynucleoside triphosphates by an enzymatic assay employing DNA polymerase I of Escherichia coli. From 2% to 0.02% O2, the free radical level decreased continually from a normal value to just above detectability by the EPR measurement employed, and quickly recovered when hypoxic cells were resupplied with atmospheric O2. Concurrently, analogous changes of the size of the dCTP pool occurred, whereas the pool sizes dATP and dGTP underwent no changes, and the size of the dTTP pool only moderate changes. The changes of the free radical concentration and of the dCTP pool correlated well with the suppression or reactivation of DNA replication under the respective O2 conditions. The results consistently support the hypothesis of a fast‐acting regulatory pathway that controls the rate of DNA replication in proliferating cells according to sufficient availability of O2. Therefore, ribonucleotide reductase may serve, in addition to providing DNA building blocks, as a PO2 sensor, which transmits the signal in the form of an altered intracellular dCTP concentration, directly or indirectly, to the nuclear‐replication machinery.
The GLY1 Gene of Saccharomyces Cerevisiae Encodes a Low‐Specific L‐threonine Aldolase that Catalyzes Cleavage of L‐allo‐Threonine and L‐threonine to Glycine
Authors: Liu Ji‐Quan , Nagata Shinji , Dairi Tohru , Misono Haruo , Shimizu Sakayu , Yamada Hideaki
Published: July 2004

The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F.‐R., Tang, S. & Bognar, A. L. (1994)J. Biol. Chem. 269, 9155–9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L‐allo‐threonine aldolase of Aeromonas jandiae DK‐39, which stereospecifically catalyzes the interconversion of L‐allo‐threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome‐binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5′‐phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L‐allo‐threonine to glycine but also L‐threonine. We have termed the enzyme a low‐specific L‐threonine aldolase to distinguish it from L‐allo‐threonine aldolase.
Ribosomal Protein S15 from Thermus Thermophilus
Authors: Serganov Alexander , Rak Alexey , Garber Maria , Reinbolt Joseph , Ehresmann Bernard , Ehresmann Chantal , Grunberg‐Manago Marianne , Portier Claude
Published: July 2004

A 6‐kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a fully repressed inducible promoter because S15 from T. thermophilus is toxic for E. coli cells. When purified without denaturation from either overproducing E. coli strain or from T. thermophilus ribosomes, the S15 protein is stable and binds a cloned T. thermophilus 16S rRNA fragment (nucleotides 559–753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for the binding of S15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E. coli S15 and T. thermophilus 16S rRNA fragment. These results suggest that S15 from T. thermophilus recognizes similar determinants in both rRNA fragments. Competition experiments support this conclusion.