bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased incorporation of arachidonic acid into phospholipids in zymosan‐stimulated mouse peritoneal macrophages
Authors: BALSINDE Jesús , FERNÁNDEZ Belén , SOLÍS‐HERRUZO José A.
Published: March 2005

Zymosan, a particle that can be phagocytosed, has been shown to stimulate the release of arachidonic acid (Δ4Ach) in macrophages via a phospholipase‐A2‐mediated mechanism, and to promote the incorporation of this fatty acid into cellular phospholipids [Balsinde, J., Fernández, B., Solís‐Herruzo, J. A. & Diez, E. (1992) Biochim. Biophys. Acta 1136, 75–82]. This work was designed to better understand the regulation of and relationship between these two processes during cellular activation. Initial studies were conducted to examine the incorporation of exogenous [3H]Δ4Ach into the different phospholipid classes. Phosphatidylcholine and phosphatidylinositol initially accounted for most of the radioactivity incorporated into the cell. Prolonged incubation resulted in a decrease in radioactivity in phosphatidylcholine with a concomitant increase in phosphatidylethanolamine. Stimulation of the cells with zymosan led to a remarkable enhancement of the response without changing the pattern of phospholipid acylation by Δ4Ach. In the next series of experiments, the regulatory features of both Δ4Ach release and phospholipid acylation by Δ4Ach in zymosan‐treated cells were comparatively investigated. Zymosan‐stimulated [3H]Δ4Ach release from previously labeled cells was notably reduced when calcium was absent from the incubation medium and also when the cells were treated with pertussis toxin. Cell treatment with cholera toxin promoted a potentiation of the response. In contrast, neither of these treatments had appreciable effects on zymosan‐stimulated [3H]Δ4Ach incorporation into phospholipids. Taken together, these data suggest that zymosan‐stimulated Δ4Ach release and phospholipid acylation by Δ4Ach, although closely related, are independently regulated events.
(Rp)‐ and (Sp)‐8‐piperidino‐adenosine 3′,5′‐(cyclic)thiophosphates discriminate completely between site A and B of the regulatory subunits of cAMP‐dependent protein kinase type I and II
Authors: ØGREID Dagfinn , DOSTMANN Wolfgang , GENIESER Hans‐Gottfried , NIEMANN Percy , DØSKELAND Stein Ove , JASTORFF Bernd
Published: March 2005

8‐Piperidino‐cAMP has been shown to bind with high affinity to site A of the regulatory subunit of cAMP‐dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme II [Øgreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Døskeland, S. O. (1989), Eur. J. Biochem. 181, 19–31]. To further increase this selectivity, the (RP)‐ and (SP)‐diastereoisomers of 8‐piperidino‐cAMP[S] were synthesized and analyzed for their potency to inhibit binding of [3H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP‐dependent protein kinases.

(SP)‐8‐Piperidino‐cAMP[S] showed an enhanced relative affinity for site AI, thus being by far the best A‐selective compound (more than 100‐fold) tested for this isozyme. In contrast, the (RP)‐ isomer was less selective for AI than 8‐Piperidino‐cAMP itself. The reduction in affinities for BII, compared to 8‐piperidino‐cAMP, were 10‐fold and 50‐fold for the (SP)‐ and (RP)‐isomer, respectively. Both isomers were almost completely excluded from AII, with affinities about 1000‐fold lower than 8‐piperidino‐cAMP itself. The (RP)‐isomer selected BII with an affinity about 10000 times higher than for AII, whereas the (SP)‐isomer showed a preference of about 70000‐fold in favour of BII. 8‐Piperidino‐cAMP as well as its (SP)‐isomer activated both types of holoenzyme protein kinases whereas the (RP)‐isomer acted as an antagonist of cAMP‐induced activation.

The study concludes that the combination of piperidino‐ and exocyclic sulfur substitutions generate cAMP analogs that completely discriminate between site A and B of cAMP‐dependent protein kinases.
Betaine counteracts urea‐induced conformational changes and uncoupling of the human erythrocyte Ca2+ pump
Authors: COELHO‐SAMPAIO Tatiana , FERREIRA Sergio T. , CASTRO Eduardo J. , VIEYRA Adalberto
Published: March 2005

We have investigated the effects of the protein structure‐perturbing and function‐perturbing osmolyte urea, and one of its physiological counteracting solutes, the methylamine compound (carboxymethyl)trimethylammonium hydroxide (betaine), on the structure and function of the human erythrocyte plasma‐membrane Ca2+‐ATPase. Betaine per se promoted a conformational change in the purified ATPase as revealed by steady‐state and time‐resolved intrinsic fluorescence spectroscopy. The conformational change promoted by betaine was shown to be related to changes in the degree of compaction of the protein structure, as detected by fluorescence‐quenching measurements using acrylamide and iodide, non‐charged and charged quenchers, respectively. In contrast, urea promoted a biphasic increase in exposure of tryptophan residues of the purified ATPase to the aqueous medium. With the use of membrane‐bound ATPase, increasing concentrations of urea up to 1.5 M promoted a twofold increase in the Ca2+‐ATPase activity, and the simultaneous inhibition of Ca2+ accumulation indicated that ATP hydrolysis became uncoupled from Ca2+ transport. Higher urea concentrations promoted a pronounced inhibition of ATP hydrolysis. In the absence of urea, betaine decreased ATP hydrolysis without affecting Ca2+ transport, whereas it counteracted the strong inhibition of Ca2+‐ATPase activity by urea concentrations as high as 7 M. Betaine also protected Ca2+ accumulation against inhibition with concentrations of urea up to 1.5 M, indicating that the methylamine is able to counteract the uncoupling of the ATPase observed at lower urea concentrations. These results suggest that betaine modifies the effects of urea on the erythrocyte Ca2+‐ATPase, through specific solute‐induced conformational changes that protect the energy‐transduction capacity of the enzyme.
A model for insulin binding to the insulin receptor
Authors: SCHÄFFER Lauge
Published: March 2005

The affinities of a number of insulin analogues for the human insulin receptor, a truncated soluble form of the insulin receptor, and the human insulin‐like growth factor 1 receptor were determined. Insulin analogues with substitutions in the A13 or B17 positions were shown to have anomalous binding properties. This suggests that these positions, which are located in the hexamer‐forming surface on the opposite side of the molecule from the classical binding site, constitute a second domain of the molecule important for receptor binding. In the present work, a model is proposed where each of the two α subunits of the insulin receptor contributes with a different binding region to the formation of the high‐affinity binding site. Subsequently, a second molecule of insulin is able to bind to a low‐affinity site involving only one of the α subunits, thus accounting for the curvilinear Scatchard plot. The affinity of the low‐affinity site could be estimated using a high‐affinity insulin analogue as the tracer. The model also provides the framework for a molecular explanation of the negative cooperativity phenomenon.
Hydrolysis of lactosylceramide by human galactosylceramidase and GM1‐β‐galactosidase in a detergent‐free system and its stimulation by sphingolipid activator proteins, sap‐B and sap‐C Activator proteins stimulate lactosylceramide hydrolysis
Authors: ZSCHOCHE Alexandra , FÜRST Werner , SCHWARZMANN Günter , SANDHOFF Konrad
Published: March 2005

Two exo‐β‐galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1‐β‐galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap‐B and sap‐C, rather than detergents, to stimulate the reaction.

While sap‐B was a better activator for the reaction catalyzed by GM1‐β‐galactosidase, sap‐C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome‐integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap‐B‐stimulated hydrolysis of liposomal ganglioside GM1 by GM1‐β‐galactosidase and sap‐C‐stimulated degradation of liposomal galactosylceramide by galactosylceramidase.

Kinetic and dilution experiments indicated that sap‐B forms water‐soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1‐β‐galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1‐β‐galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap‐C and sap‐B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.
Characterization of a polyhistidine‐tagged form of human myristoyl‐CoA:protein N‐myristoyltransferase produced in Escherichia coli
Authors: McILHINNEY R. A. Jeffrey , PATEL Paru B. , McGLONE Kate
Published: March 2005

The enzyme myristoyl‐CoA:protein N‐myristoyltransferase is responsible for the attachment of a myristoyl group to the N‐terminal glycine of a number of cell, viral and fungal proteins. In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N‐terminally polyhistidine‐tagged version of the enzyme and expressed this in Escherichia coli. The resulting enzyme has a molecular mass of 53 kDa and is fully active showing the expected specificity for myristic acid and causing the N‐terminal myristoylation of both synthetic peptide and protein substrates in vitro. The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl‐CoA of 7.6 μM. The two synthetic peptide substrates based on the N‐terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSS‐KSKPKDPSQRRRY) have different kinetic parameters with Km values of 115.2 μM and 44.2 μM and Vmax values of 95 and 120 nmol · min−1· mg−1, respectively. The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10 mM. This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor. Antisera raised to synthetic peptides based on sequences derived from the N‐ and C‐terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N‐terminal sequence reacted with the native protein suggesting that the C‐terminus may be not be accessible. The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A.
Inositol 1,4,5‐trisphosphate activates receptor‐mediated calcium entry by two different pathways in hepatocytes
Authors: STRIGGOW Frank , BOHNENSACK Ralf
Published: March 2005

The quenching of fura‐2 fluorescence by the influx of extracellular Mn2+ was measured to indicate the flux rates through receptor‐operated calcium channels in the plasma membrane of rat hepatocytes. Neomycin, an inhibitor of phospholipase C, inhibited the vasopressin‐induced influx of Mn2+. Thus, the agonist‐induced entry of extracellular calcium into hepatocytes is linked to a phospholipase C‐generated second messenger. Microinjection of inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] or 3‐deoxy‐3‐fluoro‐Ins(1,4,5)P3 revealed that Ins(1,4,5)P3 rather than Ins(1,3,4,5)P4 is responsible for calcium entry. The activation of phospholipase C by vasopressin produced an influx of Mn2+ independent of the depletion of intracellular calcium stores if this depletion was delayed by the Ins(1,4,5)P3 receptor antagonist heparin or by the use of a low agonist concentration. Thapsigargin, an inhibitor of the store calcium pump, leading to an Ins(1,4,5)P3‐independent emptying of stores, gave a short living signal (less than 3 min) for calcium entry. We propose that Ins(1,4,5)P3 is able to stimulate calcium entry by two pathways. (a) Ins(1,4,5)P3 activates receptor‐operated calcium channels in a direct manner. The calcium entry resulting from this is followed (b) by the Ins(1,4,5)P3‐induced depletion of calcium stores, producing a store‐dependent entry.
Expression in Escherichia coli of a secreted invertebrate ferritin
Authors: VON DARL Matthias , HARRISON Pauline M. , BOTTKE Werner
Published: March 2005

The coding regions of the cDNAs for cytoplasmic soma ferritin and secreted yolk ferritin from the snail Lymnaea stagnalis were inserted into the prokaryotic expression vector pEMBLex2. The vector directed the synthesis in Escherichia coli of soma ferritin up to a concentration of 15% of soluble proteins. Soma ferritin was expressed as the multimeric protein (480 kDa). Its similarity with natural soma ferritin was confirmed by PAGE, immunostaining and electron microscopy. Yolk ferritin was expressed in the form of inclusion bodies. Attempts to refold and assemble the purified yolk ferritin subunit in vitro failed. The yolk ferritin coding sequence was therefore inserted into the expression vector pMAL‐p2.

At a growth temperature of the bacterial cells of 23°C and at an isopropyl β‐d‐thiogalactopyranoside concentration of 50 μM, about 5% of the induced MalE‐yolk‐ferritin fusion protein was secreted into the periplasmic space and could be purified by affinity chromatography on amylose; the rest occurred as insoluble cytoplasmic inclusion bodies. Soluble MalE–yolk‐ferritin fusion protein was capable of assembly into ferritin‐like particles. Fully assembled yolk apoferritin shells (610 kDa) were obtained by digestion of these particles with proteinase K (yield: 180 μg yolk ferritin/l bacterial culture). Recombinant yolk ferritin was capable of taking up iron in vitro. Yolk ferritin (610 kDa) and soma ferritin (480 kDa) were run to the pore limit of a non‐denaturing 5–20% PAGE gradient gel. Under these conditions, yolk ferritin had a higher mobility than soma ferritin (480 kDa) and therefore the yolk ferritin may have a rather compact structure. A 41‐amino‐acid‐residue stretch of the insertion, a distinctive feature of the yolk ferritin subunit, was deleted by site‐directed mutagenesis. The MalE–yolk‐ferritin variant thus obtained was readily degradable by proteinase K and could not be assembled into ferritin‐like particles. Therefore residues in the deleted peptide must be important for the maintenance of the native structure.
Bovine inositol monophosphatase
Authors: GREASLEY Peter J. , HUNT Lawrence G. , GORE Michael G.
Published: March 2005

Inositol monophosphatase can be modified at two sites by pyrene maleimide. These sites have been identified as Cys141 and Cys218. Stoichiometric addition of pyrene maleimide allows the sole modification of Cys218. The fluorescence of the pyrene moiety on the modified protein can be excited directly or by resonance energy transfer. The fluorescence properties of the pyrene group on Cys218 allows the interaction of ligands with the enzyme to be monitored. This feature has allowed dissociation constants for various metal ions to be determined and allowed the formation of various enzyme/ligand complexes to be observed. These studies have demonstrated that Mg2+ is required to support Pi binding and that Li+ interacts with a post‐catalytic complex which is only formed in the forward reaction.
Neutralizing monoclonal antibodies define two different functional sites in human interleukin‐4
Authors: REUSCH Petra , ARNOLD Stefan , HEUSSER Christoph , WAGNER Kathrin , WESTON Beverly , SEBALD Walter
Published: March 2005

Human interleukin‐4 (IL‐4) is a small four‐helix‐bundle protein which is essential for organizing defense reactions against macroparasites, in particular helminths. Human IL‐4 also appears to exert a pathophysiological role during various IgE‐mediated allergic diseases. Seven different monoclonal antibodies neutralizing the activity of human IL‐4 were studied in order to identify functionally important epitopes. A collection of 41 purified IL‐4 variants was used to analyse how defined amino acid replacements affect binding affinity for each individual mAb. Specific amino acid positions could be assigned to four different epitopes. mAbs recognizing epitopes on helix A and/or C interfered with IL‐4 receptor binding and thus inhibited IL‐4 function. However, other mAbs also inhibiting IL‐4 function recognized an epitope on helix D of IL‐4 and did not inhibit IL‐4 binding to the receptor protein. One mAb, recognizing N‐terminal and C‐terminal residues, partially competed for binding to the receptor. The results of these mAb epitope analyses confirm and extend previous data on the functional consequences of the amino acid replacements which showed that amino acid residues in helices A and C of IL‐4 provide a binding site for the cloned IL‐4 receptor and that a signalling site in helix D interacts with a further receptor protein.
Calcium release activity and metabolism of inositol 1,4,5‐trisphosphate in T cells
Authors: WARD Stephen G. , LAMPE Dethard , LIU Changsheng , POTTER Barry V. L. , WESTWICK John
Published: March 2005

Stimulation of the T cell antigen receptor/CD3 complex is followed by phospholipase C activation, phosphoinositol lipid metabolism and ultimately by a rapid rise in both myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and myo‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] as well as cytosolic free calcium concentration. A 5‐phosphatase plays a pivotal role in the subsequent metabolism of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Synthetic routes have been developed which have enabled the synthesis of both natural and unnatural inositol phosphates and this approach has yielded several compounds which have been shown to act as inhibitors of Ins(1,4,5)P3 5‐phosphatase. These compounds offer considerable potential for investigation of the complex metabolism and function of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in T cell activation and proliferation. We now report the time course and temperature sensitivity of Ins(1,4,5)P3‐induced 45Ca2+ release in the permeabilised leukaemic T cell line Jurkat. Furthermore, we demonstrate that the metabolism of Ins(1,4,5)P3 in the presence of two novel 5‐phosphatase inhibitors, namely L‐myo‐inositol 1,4,5‐trisphosphorothioate [L‐Ins(1,4,5)PS3] and myo‐inositol 1,3,5‐trisphosphorothioate [Ins(1,3,5)PS3], can be inhibited with concomitant elevation of the heparin‐sensitive Ins(1,4,5)P3‐induced release of 45Ca2+. These novel 5‐phosphatase inhibitors provide a starting point for development of cell‐permeable analogues which may be able to modulate cell function in intact cells and may be used as manipulative tools with which to elucidate the function of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 with respect to T cell activation.
Glucokinase expression in rat hepatoma cells induces glucose uptake and is rate limiting in glucose utilization
Authors: VALERA Alfons , BOSCH Fatima
Published: March 2005

In contrast to hepatocytes, hepatoma cells lack glucokinase activity and show increased aerobic glycolysis. FTO‐2B and H4IIE rat hepatoma cell lines were obtained in which the rat glucokinase gene was expressed (FTOGK and H4GK). These lines were generated by infection of the hepatoma cells with a retroviral vector carrying the phosphoenolpyruvate carboxykinase (PEPCK)‐glucokinase chimeric gene. Both the FTOGK and H4GK cells expressed the chimeric gene in a regulated manner, like the endogenous PEPCK gene. Glucokinase activity was detected in both FTOGK and H4GK. These cell lines showed a marked increase in glucose uptake with 18.5 mM glucose in the incubation medium. FTOGK and H4GK showed an increase in the content of glucose 6‐phosphate, and were able to accumulate high levels of glycogen, in contrast to FTO‐2B cells, which were unable to store the polysaccharide. In addition, cells expressing glucokinase showed high concentration of fructose 2,6‐bisphosphate and substantial lactate production, which was related to the glucose concentration in the medium and the time of incubation.

These results suggest that glucose phosphorylation is rate limiting for glucose uptake and utilization in FTO‐2B and H4IIE cells.
Isolation, characterization and expression of cDNAs encoding the catfish‐type and chicken‐II‐type gonadotropin‐releasing‐hormone precursors in the African catfish
Authors: BOGERD Jan , ZANDBERGEN Thijs , ANDERSSON Eva , GOOS Henk
Published: March 2005

The cDNAs encoding the catfish prepro‐gonadotropin‐releasing hormone and the chicken pre‐pro‐gonadotropin‐releasing hormone II of the African catfish (Clarias gariepinus) have been isolated and sequenced. The catfish gonadotropin‐releasing‐hormone precursor and the chicken gona‐dotropin‐releasing‐hormone‐II precursor have the same overall architecture as other gonadotropin‐releasing‐hormone precursors identified so far; each is composed of a signal peptide, gonadotropin‐releasing hormone and a gonadotropin‐releasing‐hormone‐associated peptide which is connected to gonadotropin‐releasing hormone by a Gly‐Lys‐Arg sequence. The amino acid sequences of catfish gonadotropin‐releasing hormone and chicken gonadotropin‐releasing hormone II, in combination with the Gly‐Lys‐Arg sequence, are highly conserved during evolution when compared with the corresponding regions of mammalian, avian (chicken gonadotropin‐releasing hormone I) and other fish gonadotropin‐releasing‐hormone precursors. However, the gonadotropin‐releasing‐hormone‐as‐sociated peptide regions are markedly divergent. Northern‐blot analysis revealed the presence of a single catfish gonadotropin‐releasing‐hormone mRNA species of about 470 bases, and the presence of a single chicken gonadotropin‐releasing‐hormone‐II mRNA species of about 650 bases in the African catfish brain. In situ hybridization revealed catfish gonadotropin‐releasing‐hormone cell bodies rostro‐caudally scattered in the olfactory nerve, along both sides of the midline of the telencephalon, in the preoptic area of the ventral hypothalamus, and in the infundibular stalk close to the pituitary. Chicken gonadotropin‐releasing‐hormone‐II cell bodies, however, were exclusively found in the midbrain tegmentum.
Construction, expression and characterization of a plasmid‐encoded Na+‐specific ATPase hybrid consisting of Propionigenium modestum F0‐ATPase and Escherichia coli F1‐ATPase
Authors: KAIM Georg , DIMROTH Peter
Published: March 2005

The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, δ and part of the α subunit of the Na+‐dependent ATPase of Propionigenium modestum and the genes encoding the α, γ, β and ε subunits of the H+‐dependent E. coli ATPase. The transformants showed Na−‐dependent growth on succinate as non‐fermentable carbon source. The functionally expressed hybrid ATPase was activated 13‐fold at pH 7.5 by the addition of Na+ and inhibited by 1,3‐dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3‐dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP‐dependent fluorescence quenching of 9‐amino‐6‐chloro‐2‐methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin‐mediated K+ diffusion potential (Ψ) and a Na+ concentration gradient (pNa+) were imposed.
Optical spectrum of myeloperoxidase
Authors: FLORIS René , KIM Younkyoo , BABCOCK Gerald T. , WEVER Ron
Published: March 2005

The optical spectrum of reduced myeloperoxidase (EC 1.11.1.7) displays an unusual red shift of the Soret band which is at 472 nm and the α‐band which is at 636 nm. The spectral properties of myeloperoxidase can be modified by means of acid treatment. Upon short exposure to acid (pH 1.7) the red‐shifted optical absorption spectrum of the reduced enzyme (λmax at 472 nm) was blue‐shifted (λmax at 448 nm) but the spectrum of the reduced state could be restored by increasing the pH. By contrast, the resonance Raman spectra of both the oxidized and reduced enzyme are essentially the same at both pH 1.7 and pH 7.0. This shows that the optical spectrum and the resonance Raman spectrum are not directly correlated, which we interpret to indicate that the reversible effects of lower pH primarily affect the excited‐state energy levels of the macrocycle. The EPR spectrum of the oxidized enzyme showed a reversible conversion from a high‐spin rhombic spectrum (gx= 6.7, gy= 5.2) at neutral pH into a more axial high‐spin spectrum (gx=gy= 5.8) at low pH.

Upon prolonged exposure to acid (20 min) optical absorbance spectra, EPR spectra, resonance Raman spectra and the chlorinating activity were irreversibly affected. We propose that a negatively charged protonatable residue in the proximity of a pyrrole nucleus of the haem group is present that imposes the red shift in the optical absorption spectrum. This is consistent with the available X‐ray structure data.
Isolation of protein FA, a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli
Authors: PALMER Tracy , VASISHTA Anil , WHITTY Patrick W. , BOXER David H.
Published: March 2005

The mob mutants in Escherichia coli are pleiotropically defective in all molybdoenzyme activities. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (protein factor d'association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30‐fold more protein FA activity than the wild‐type bacterium. Protein FA has been purified to homogeneity by a combination of ion‐exchange, affinity and gel‐filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N‐terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation.
Post‐translational modifications in aspartate aminotransferase from Sulfolobus solfataricus
Authors: ZAPPACOSTA Francesca , SANNIA Giovanni , SAVOY Luc‐Alain , MARINO Gennaro , PUCCI Piero
Published: March 2005

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments.

The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast‐atom‐bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N‐ɛ‐methyl derivatives to a substoichiometric extent.
Characterization of acyl‐CoA thioesterase activity in isolated rat liver peroxisomes
Authors: WILCKE Mona , ALEXSON Stefan E. H.
Published: March 2005

A common function of peroxisomes in eukaryotic cells is β‐oxidation of fatty acids. In animal cells, β‐oxidation is compartmentalized to peroxisomes and mitochondria. Although regulation of β‐oxidation in mitochondria has been extensively studied, knowledge on its regulation in peroxisomes is still limited. We have considered the possibility that peroxisomes may contain acyl‐CoA thioesterases with different substrate specificities that possibly regulate metabolism of different lipids by regulation of substrate availability. In the present study, we have investigated the presence of short‐chain and long‐chain acyl‐CoA thioesterase activities in rat liver peroxisomes.

Light‐mitochondrial fractions, enriched in peroxisomes, were fractionated by Nycodenz density gradient centrifugation and gradient fractions were analyzed for acyl‐CoA thioesterase and marker enzyme distributions. Fractionation of livers from normal rats showed that most of the long‐chain acyl‐CoA thioesterase activity was localized in microsomes and mitochondria, and only low activity was found in fractions containing peroxisomes. The gradient distribution of propionyl‐CoA thioesterase activity showed this activity to be localized mainly in mitochondria and in fractions possibly representing lysosomes, with a small peak of activity in peroxisomal fractions.

Di(2‐ethylhexyl)phthalate treatment induced the specific propionyl‐CoA thioesterase activity approximately threefold in the peak mitochondrial fractions and about onefold in peroxisomal fractions; the activity appeared to be almost exclusively localized to these organelles. The specific activity of myristoyl‐CoA thioesterase was induced 1–2‐fold in peroxisomal peak fractions and more than 10‐fold in the mitochondrial peak fraction, whereas it was unchanged in microsomes.

The chain‐length specificity of acyl‐CoA thioesterase activity in isolated peroxisomes suggests that peroxisomes contain an inducible short‐chain thioesterase active on C2‐C4 acyl‐CoA species (possibly a ‘propionyl‐CoA’ thioesterase). In addition, peroxisomes contain medium‐chain to long‐chain thioesterase activity, probably due to separate enzymes based on the different chain‐length specificities observed in peroxisomes from normal and di(2‐ethylhexyl)phthalate‐treated rats.

A long‐chain acyl‐CoA thioesterase was partially purified from isolated peroxisomes and found to be active only on fatty‐acyl‐CoA species longer than octanoyl‐CoA. The protein is apparently a monomer of about 40 kDa and clearly different from microsomal long‐chain acyl‐CoA thioesterase. An induction of this long‐chain thioesterase may explain the observed change in chain‐length specificity in peroxisomes isolated from normal and di(2‐ethylhexyl)phthalate‐treated rats.

Possible physiological functions of these thioesterases are discussed.
Sequencing and characterization of the sdaC gene and identification of the sdaCB operon in Escherichia coli K12
Authors: SHAO ZhongQi , LIN Rong Tuan , NEWMAN E. B.
Published: March 2005

We describe here the regulatory and coding region, and DNA sequence, for a newly recognized gene, sdaC, which codes for a hydrophobic protein with several predicted membrane‐spanning domains. sdaC and sdaB form a single operon, with 57 bp between the end of sdaC and the start of sdaB. Expression of the sdaCB operon is regulated mainly by catabolite repression, but is also slightly sensitive to regulation by leucine‐responsive regulatory protein. Cells carrying sdaC on a multicopy plasmid have increased L‐serine transport capacity, insensitive to threonine, suggesting that sdaC might code for a recently described highly specific serine transporter [Kayahara, T., Thelen, P., Ogawa, W., Inaba, K., Tsuda, M., Goldberg, E. B. & Tsuchiya, T. (1992) J. Bacteriol. 174, 7482–7485].
Purification of a bone sialoprotein‐binding protein from Staphylococcus aureus
Authors: YACOUB Alia , LINDAHL Per , RUBIN Kristofer , WENDEL Mikael , HEINEGÅRD Dick , RYDÉN Cecilia
Published: March 2005

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C., Maxe, I., Franzén, A., Ljungh, Å., Heinegård, D. & Rubin, K. (1987) Lancet II, 515]. We now report on the purification of a cell‐wall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell‐wall components with capacity to inhibit binding of 125I‐labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0).

Preparative SDS/PAGE and protein‐overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with Mr 75000–110000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono‐Q anion‐exchange column. Inhibitory activity was eluted at 0.6–0.8 M NaCl and could be further purified by affinity chromatography on BSP‐Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity.

Analysis by SDS/PAGE revealed a single Mr 97000 polypeptide in the eluate. The purified Mr 97000 protein bound BSP in protein‐overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the Mr 97000 protein. Our data demonstrate the presence of a S. aureus, cell‐surface BSP‐binding protein. This protein could be involved in bacterial tropism in osteomyelitis.
ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state
Authors: MEDINA Milagros , VRIELINK Alice , CAMMACK Richard
Published: March 2005

Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone. The rate and extent of reduction were pH‐dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M−1 cm−1. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X‐band or at S‐band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 9–12 MHz, is attributed to protons on 8‐CH3 (Aiso= 10.9MHz) and on C6 (Aiso= 9 MHz) of the flavin ring. No change in these hyperfine coupling constants was detected when the protein was studied in D2O. However, the hyperfine coupling constant attributed to protons on 8‐CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso= 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz. Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present. These signals are attributed to displaced water protons, or to exchangeable protons from amino‐acid residues on the protein near the flavin binding site, involved in substrate stabilisation.
Ubisemiquinones as obligatory intermediates in the electron transfer from NADH to ubiquinone
Authors: DE JONG A. Mariette P. , ALBRACHT Simon P. J.
Published: March 2005

Until now ubisemiquinones associated with NADH: ubiquinone oxidoreductase (complex I) have been reported to occur in isolated enzyme and in tightly coupled submitochondrial particles. In this report it is shown that ubisemiquinones are always detectable during steady‐state electron transfer from NADH to ubiquinone, independent of the type of inner‐membrane preparation used. The EPR signal of the rotenone‐sensitive ubisemiquinones could be detected not only in coupled MgATP submitochondrial particles, but also in routine preparations of uncoupled submitochondrial particles and in mitochondria. The ubisemiquinone formation in coupled preparations was completely insensitive to uncouplers. The maximal radical concentration during steady‐state electron transfer from NADH to quinone was equal to that of iron‐sulphur cluster 2. Experiments with antimycin, myxothiazol and 2‐thenoyltrifluoroacetone demonstrated that about half of this radical was associated with complex I, giving a ubisemiquinone concentration of about 0.5 mol semiquinone/mol cluster 2. Uncoupled submitochondrial particles, prepared by extensive sonification, never showed radical signals within 100 ms after mixing with NADH. This was due to the reversible inactivation of the enzyme, caused by elevated temperatures during sonification. In preparations with deliberately heat‐inactivated complex I, no radical signals were detected within 200 ms after mixing with NADH; at 1 s, however, radical formation was maximal. Yet, depending on the procedure of reactivation of the complex, in preparations previously treated to inactivate them ubisemiquinone concentrations were always less than in untreated particles. When complex I was in the active state the ubisemiquinone signal was maximal within 40 ms. The results described in this report lead to the conclusion that ubisemiquinones form obligatory intermediates in the reaction of NADH dehydrogenase with ubiquinone.
The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria
Authors: ROBINSON Karen M. , ROTHERY Richard A. , WEINER Joel H. , LEMIRE Bernard D.
Published: March 2005

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron‐transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron‐sulfur protein subunit with three different iron‐sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8α‐[N(3)‐histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non‐covalently. The resulting holoenzyme has no succinate‐dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.
Reversible membrane association of heat‐shock protein 22 in Chlamydomonas reinhardtii during heat shock and recovery
Authors: EISENBERG‐DOMOVICH Yael , KLOPPSTECH Klaus , OHAD Itzhak
Published: March 2005

The process of reversible membrane association of the nuclear‐encoded heat‐shock protein hsp22 in Chlamydomonas reinhardtii cells during recovery from heat stress has been investigated. hsp22 associates with a chloroplast membrane‐enriched fraction, dissociates from the membranes during recovery from heat shock and rebinds during a subsequent heat‐shock treatment in vivo. The protein remains in the cell soluble fraction for at least 22 h after heat‐stress treatment. Dissociation of membrane‐bound hsp22 occurs only at 25–38°C and reassociation occurs only at the hsp22 induction temperature (38–42°C). Hsp22 dissociation from the membrane fraction is not related to de novo protein synthesis in vivo and does not occur in vitro. Based on the derived amino acid sequence, hsp22 is not considered a typical chloroplast‐associated heat‐shock protein [Vierling, E. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 579–620] and may be associated with the chloroplast envelope membrane. However, the reversible association of hsp22 with the chloroplast‐enriched membrane fraction indicates similar properties to those of pea low‐molecular‐mass heat‐shock proteins [Glaczinski, H. & Kloppstech, K. (1988) Eur. J. Biochem. 173, 579–583] and may be related to the transient response of the chloroplast to heat stress.
Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n‐octyl β‐d‐glucoside
Authors: YOSHIDA Mikiharu , SUZUKI Atsushi , YAMAMOTO Hideko , NOGUCHI Satoru , MIZUNO Yuji , OZAWA Eijiro
Published: March 2005

Dystrophin is purified as a complex with several proteins from the digitonin‐solubilized muscle cell membrane. Most of dystrophin‐associated proteins (DAPs) are assumed to form a large oligomeric transmembranous glycoprotein complex on the sarcolemma and link dystrophin with a basement membrane protein, laminin. In the present study, we found that the purified dystrophin‐DAP complex was dissociated into several groups by n‐octyl‐β‐d‐glucoside treatment. In particular, we found that the glycoprotein complex stated above was dissociated into two distinct groups: one composed of 156DAG and 43DAG (A3a) and the other composed of 50DAG, 35DAG and A3b. We confirmed by crosslinking and immunoaffinity chromatography that these two groups existed in a complexes. We thus concluded that the glycoprotein complex consists of these two subcomplexes. Furthermore, A3b and 43DAG, which had been formerly treated simply as the 43DAG doublets due to their similar electrophoretic mobilities in SDS/PAGE, were shown to be present in two different subcomplexes. Based on the analyses by two‐dimensional gel electrophoresis, peptide mapping and immunoblotting, we concluded that A3b is a novel DAP different from 43DAG.
Mobile ionophores are a novel class of P‐glycoprotein inhibitors
Authors: BORREL Marie Nicole , PEREIRA Elene , FIALLO Marina , GARNIER‐SUILLEROT Arlette
Published: March 2005

The decrease of the intracellular concentration of drug in resistant cells compared to sensitive cells is, in most cases, correlated with the presence, in the membrane of resistant cells, of a 170‐kDa P‐glycoprotein responsible for an active efflux of the drug. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, we have examined the cellular accumulation of 4′‐O‐tetrahydropyranyl‐adriamycin, alone and in conjunction with various ionophores on the one hand and with cyclosporin A on the other hand. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. Using experimental conditions in which these ionophores were unable to modify either the intracellular pH, or the transmembrane potential, or to induce an intracellular ATP depletion, we have shown that mobile ionophores as well as cyclosporin inhibit the P‐glycoprotein‐mediated efflux of 4′‐O‐tetrahydropyranyl‐adriamycin in K562 resistant cells, whereas gramicidin, a channel‐forming ionophore, does not. The concentration that must be used to inhibit 50% of the efflux was 0.7 μM for valinomycin, 0.4 μM for nonactin, 0.2 μM for nigericin, 1.1 μM for monensin, 0.4 μM for lasalocid, 1.2 μM for calcimycin and 0.4 μM for cyclosporin. Due to the high toxicity of the ionophores, the observation that they increased 4′‐O‐tetrahydropyranyl‐adriamycin accumulation in the multidrug‐resistant cells is not correlated with an effect of these compounds on drug resistance. However, the correlation exists in the case of cyclosporin. From our data showing that lipophilic neutral complexes, formed between carboxylic ionophores and metal ions, as well as lipophilic cationic complexes, formed between neutral ionophores and metal ions, are both able to inhibit the P‐glycoprotein‐mediated efflux of anthracycline we can infer that the lipophilicity but not the cationic charge is an important physical property.
Pseudothionin‐St1, a potato peptide active against potato pathogens
Authors: MORENO Manuel , SEGURA Ana , GARCÍA‐OLMEDO Francisco
Published: March 2005

A 5‐kDa polypeptide, pseudothionin Solanum tuberosum 1(Pth‐St1), which was active against Clavibacter michiganensis subspecies sepedonicus, a bacterial pathogen of potatoes, has been purified from the buffer‐insoluble fraction of potato tubers by salt extraction and HPCL. Pth‐St1 was also active against other potato pathogens tested (Pseudomonas solanacearum and Fusarium solani). The N‐terminal amino acid sequence of this peptide was identical (except for a N/H substitution at position 2) to that deduced from a previously reported cDNA sequence (EMBL accession number X‐13180), which had been misclassified as a Bowman‐Birk protease inhibitor. Pth‐St1 did not inhibit either trypsin or insect α‐amylase activities, and, in contrast with true thionins, did not affect cell‐free protein synthesis or β‐glucuronidase activity. Northern‐blot and tissue‐print analyses showed that steady‐state mRNA levels were highest in flowers (especially in petals), followed by tubers (especially in the epidermal cell layers and in leaf primordia), stems and leaves. Infection of leaves with a bacterial pathogen suspended in 10 mM MgCl2 switched off the gene, whereas mock inoculation with 10 mM MgCl2 alone induced higher mRNA levels.