bluuebunny/crossref_metadata_2025 huggingface dataset:
Association with B‐cell‐antigen receptor with protein‐tyrosine kinase p72syk and activation by engagement of membrane IgM
Authors: YAMADA Takechiyo , TANIGUCHI Takanobu , YANG Cheng , YASUE Satoshi , SAITO Hitoshi , YAMAMURA Hirohei
Published: March 2005

We have demonstrated that a 72‐kDa non‐receptor‐type protein‐tyrosine kinase (p72syk) was coimmunoprecipitated with membrane IgM in digitonin lysates of porcine tonsillar cells and was rapidly activated following the engagement of membrane IgM. This activation was occurred within 5 s, even in the presence of EGTA and 5,5′‐dimethyl‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid as extracellular and intracellular Ca2+‐chelating agents, respectively, as well as in the presence of the protein‐kinase‐C inhibitor, H‐7. Additionally, genistein, a potent protein‐tyrosine kinase inhibitor, was capable of reducing both IgM‐stimulated Ca2+ mobilization and p72syk activation in a dose‐dependent manner. These results indicate that p72syk is physically associated with the B‐cell‐antigen receptor, participating in antigen‐mediated signal transduction in both a Ca2+‐independent and protein‐kinase‐C‐independent manners.
Purification of a cytochrome b containing H2:heterodisulfide oxidoreductase complex from membranes of Methanosarcina barkeri
Authors: HEIDEN Stefanie , HEDDERICH Reiner , SETZKE Edgar , THAUER Rudolf K.
Published: March 2005

The reduction of CoM‐S‐S‐HTP, the heterodisulfide of coenzyme M (H‐S‐CoM) and N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP), with H2 is an energy‐conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane‐bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non‐heme iron and acidlabile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme‐derived peroxidase activity indicating that this polypeptide is the cytochrome b.

The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM‐S‐S‐HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 μmol · min−1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM‐S‐S‐HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM‐S‐S‐HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM‐S‐S‐HTP and re‐reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions.

The findings suggest that the H2: heterodisulfide oxidoreductase complex is composed of a F420‐non‐reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM‐S‐S‐HTP reduction with H2.
Enzymes of anaerobic metabolism of phenolic compounds
Authors: BRACKMANN Ruth , FUCHS Georg
Published: March 2005

The reductive removal of aromatic hydroxyl functions plays an important role in the anaerobic metabolism of many phenolic compounds. We describe a new enzyme from a denitrifying Pseudomonas sp., 4‐hydroxybenzoyl‐CoA reductase (dehydroxylating), which reductively dehydroxylates 4‐hydroxybenzoyl‐CoA to benzoyl‐CoA. The enzyme plays a role in the anaerobic degradation of phenol, 4‐hydroxybenzoate, p‐cresol, 4‐hydroxyphenylacetate, and other aromatic compounds of which 4‐hydroxybenzoyl‐CoA is an intermediate. The enzyme is therefore induced only under anoxic conditions with these aromatic substrates, but not with benzoate or under aerobic conditions. A similar enzyme which reductively dehydroxylates 3‐hydroxybenzoyl‐CoA is induced during anaerobic growth with 3‐hydroxybenzoate. The soluble enzyme 4‐hydroxybenzoyl‐CoA reductase was purified. It has a molecular mass of 260 kDa and consists of three subunits of 75, 35, and 17 kDa. The subunit composition is likely to be a2b2c2. The enzyme contains 12 mol iron/mol and 12 mol acid‐labile sulfur/mol and exhibits a typical ultraviolet/visible spectrum of an iron‐sulfur protein. The reaction requires a reduced electron donor such as reduced viologen dyes; no other co‐catalysts are required, the product is benzoyl‐CoA and oxidized dye. The reductase is rapidly inactivated by oxygen. The inactivation by low concentrations of cyanide or azide in a pseudo‐first‐order time course suggests that it may contain a transition metal in an oxidation state which reacts with these ligands. 4‐Hydroxybenzoyl‐CoA reductase represents a type of enzyme which is common in anaerobic aromatic metabolism of phenolic compounds. A similar enzyme is demonstrated in Rhodopseudomonas palustris anaerobically grown with 4‐hydroxybenzoate. The biological significance of reductive dehydroxylation of aromatics and a possible reaction mechanism similar to the Birch reduction are discussed.
Identification of charge‐transfer transitions in the optical spectrum of low‐spin ferric cytochrome P‐450 Bacillus megaterium
Authors: McKNIGHT J. , CHEESMAN M. R. , THOMSON A. J. , MILES J. S. , MUNRO A. W.
Published: March 2005

The optical, low temperature magnetic circular dichroism (MCD) and EPR spectra of low‐spin Fe(III) cytochrome P‐450 BM‐3 from Bacillus megaterium, and its imidazole adduct have been measured. The MCD spectra locate new transitions over 600–700 am and 800–1300 nm. The latter are assigned to porphyrin (a1u)–Fe(III) (dyz) charge‐transfer (CT) transitions. In the case of the imidazole adduct the energy of this transition fits well to the theoretical model of Gadsby and Thomson [Gadsby, P. M. A. & Thomson, A. J. (1990) J. Amer. Chem. Soc 112, 5003–5011]. For the native enzyme, the energy of the CT band suggests co‐ordination by a strongly H‐bonded water ligand and the axial thiolate form of cysteine. Two transitions between 600–700 nm are detected in both derivatives. A theoretical analysis and fit of the MCD magnetisation properties shows that these transitions are polarised XY and XZ, respectively. They are assigned as thiolate sulphur py–d‐shell and pz–d‐shell CT transitions, analogous to the well known 695 nm band of methioninehistidine co‐ordinated haem as in cytochrome c. They should prove usefully diagnostic of cysteine/Fe(III) conformational changes or protonation.
Identification of a new family of human epithelial protein kinases containing two leucine/isoleucine‐zipper domains
Authors: DOROW Donna S. , DEVEREUX Lisa , DIETZSCH Erin , DE KRETSER Theonne
Published: March 2005

Using the polymerase chain reaction to study mRNA expressed in human epithelial tumor cells, a member of a new family of protein kinases was identified. The catalytic domain of this kinase has amino‐acid‐sequence similarity to both the Tyr‐specific and the Ser/Thr‐specific kinase classes. Clones representing two members of this new family have been isolated from a human colonic epithelial cDNA library and sequenced. The predicted amino‐acid sequences of these clones reveal that, in addition to the unusual nature of their kinase catalytic domains, they contain two Leu/Ilezipper motifs and a basic sequence, near their C‐termini. As they possess domains associated with proteins from two distinct functional groups, these kinases have been named mixed‐lineage kinases (MLK) 1 and 2. mRNA from MLK1 has been found to be expressed in epithelial tumor cell lines of colonic, breast and esophageal origin. The MLK1 gene has been mapped to human chromosome 14q24.3–31.
Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor
Authors: HOWL John , WANG Xianghong , KIRK Christopher J. , WHEATLEY Mark
Published: March 2005

We have designed and synthesized a linear peptide analogue of arginine vasopressin. This peptide, [1‐phenylacetyl, 2‐O‐methyl‐D‐tyrosine, 6‐arginine, 8‐arginine, 9‐lysinamide]vasopressin (PhAcALVP), has a lysinamide residue substituted for the more usual glycinamide at position 9. Derivatization of PhAcALVP at the Nɛ‐lysyl amino group with N‐hydroxysuccinimide esters of aminomethylcoumarin (Mec) and biotin (Btn) produced the bifunctional ligands PhAcAL(Mec)VP and PhAcAL(Btn)VP, respectively. Pharmacological characterization of these peptides revealed that all were high‐affinity V1a‐selective antagonists. PhAcAL(Btn)VP can simultaneously bind to both the rat liver V1a receptor and avidin conjugates. Using this strategy, we were able to study the distribution of V1a receptors on the surface of the rat mammary tumour cell line, WRK‐1. Routine epifluorescent microscopy and confocal image analysis were used to observe the distribution of avidin—Texas‐Red associated with receptor‐bound PhAcAL(Btn)VP. We conclude that PhAcALVP is a useful precursor for the production of hetero‐bifunctional V1a‐selective ligands. Both PhAcAL‐(Mec)VP and PheAcAL(Btn)VP can be used selectively to probe the V1a receptor and will be versatile tools for a variety of histocytochemical applications, including receptor localization and purification.
Conversion of fructose to glucose in the rabbit small intestine
Authors: BISMUT Hervé , HERS Henri‐Géry , Van SCHAFTINGEN Emile
Published: March 2005

Gopher et al [Gopher, A., Vaisman, N., Mandel, H. & Lapidot, A. (1990) Proc. Natl Acad. Sci. USA 87, 5449–5453] recently reported that about 50% of the glucose formed from [U‐13C]fructose infused nasogastrically in children contained 13C3 adjacent to 13C4. Assuming a high isotopic dilution of the triosephosphate pool, the authors concluded that about 50% of the fructose converted to glucose in liver and intestine bypassed the classical aldolase pathway, utilizing a hypothetical direct pathway that would involve the phosphorylation of fructose 1‐phosphate to fructose 1,6‐bisphosphate. The present work was undertaken in order to establish to what extent the conversion of fructose to glucose in the intestine could account for this unexpected isotopic distribution. The technique of everted sleeves was used to define the rate of conversion of [U‐14C]glucose and [U‐14C]fructose in the small intestine of 24‐h‐fasted rabbits. It appeared that, at the low concentration of fructose used by Gopher et al., almost as much fructose was converted to glucose as remained unmodified in the tissue. Fractose uptake was not inhibited by glucose, and the presence of all the necessary enzymes in the tissue indicated that the fructose to glucose conversion occurred by the aldolase pathway. Remarkably, this conversion operated with an isotopic dilution not exceeding 25%, due to the low rate of glucose metabolism and the near absence of gluconeogenesis from lactate. It can, therefore, be postulated that, in the presence of pure [U13C]fructose, the triosephosphate pool is highly enriched in 13C with little dilution by 12C, essentially giving rise to [U‐13C]glucose, as reported by Gopher et al. There is, therefore, no need to postulate the participation of a direct pathway.
Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution
Authors: TEPLYAKOV Alexei , OBMOLOVA Galya , WILSON Keith , KUROMIZU Kenji
Published: March 2005

The three‐dimensional structure of apo‐neocarzinostatin, an antitumour antibiotic protein isolated from Streptomyces carzinostaticus, has been determined by X‐ray diffraction at 0.15‐nm resolution and refined to R= 17.2%. The crystal structure of neocarzinostatin is similar to that of the related proteins actinoxanthin and macromomycin. It is also in good agreement with the solution structure determined by NMR spectroscopy. The protein molecule consists of a seven‐stranded antiparallel β‐sandwich and a smaller lobe formed by two β‐ribbons. A deep cleft between the two lobes is a putative chromophore binding site. Side chains of Trp39, Leu45, Phe52, Phe78 and the disulphide Cys37‐Cys47 aligning the binding cleft in neocarzinostatin suggest the importance of hydrophobic interactions in stabilizing the chromophore molecule. Comparison of the atomic models of neocarzinostatin, actinoxanthin and macromomycin reveals functional residues which might determine specificity towards different chromophores.
Molecular basis for heme‐dependent induction of heme oxygenase in primary cultures of chick embryo hepatocytes
Authors: SRIVASTAVA Kishore K. , CABLE Edward E. , DONOHUE Susan E. , BONKOVSKY Herbert L.
Published: March 2005

The effects of heme on the induction of mRNA and protein synthesis for heme oxygenase‐1 have been studied in primary cultures of chick embryo liver cells. Heme increased the amount of mRNA and the rate of heme oxygenase‐1‐gene transcription in a dose‐dependent fashion, with a maximal 20‐fold increase occurring at 20 μM heme. The largest increase in the rate of transcription, measured by nuclear run‐on assays, occurred at 5 h, 2 h earlier than the maximum increase in the amount of mRNA, measured by densitometry of Northern blots. 7–15 h after heme addition, the half‐life of heme‐oxygenase‐1 mRNA was 3.5 h in the presence or absence of actinomycin D. In contrast, addition of cycloheximide markedly increased the stability of the message (half‐life = 18 h), suggesting that a short‐lived protein plays a key role in modulating heme oxygenase‐1 mRNA levels. The half‐life of heme‐induced heme‐oxygenase‐1 protein, measured by [35S]methionine labelling and immunoprecipitation, was 15 h. This long half‐life of the protein can largely account for the additional finding that, following addition of heme, the amount of enzyme protein in the cells increased for 10 h, after which it remained essentially constant for 15 h. A striking finding was that, after an initial burst of heme‐stimulated gene transcription, the cells became refractory to further heme‐mediated induction. This acquired resistance could not be attributed to the following: a longer duration of culture time; cellular toxicity caused by heme; a lack of heme in the medium or the cells; secretion of heme‐binding proteins into the medium, preventing further heme uptake; the induction of cellular heme catabolism sufficient to deplete cellular heme. Instead, the results suggest a down‐regulation of the intracellular machinery required for heme‐dependent induction of heme oxygenase‐1.
Structure/activity relationship of adenine‐modified NAD derivatives with respect to porcine heart lactate dehydrogenase isozyme H4 simulated with molecular mechanics
Authors: HENDLE Jörg , BÜCKMANN Andreas F. , AEHLE Wolfgang , SCHOMBURG Dietmar , SCHMID Rolf D.
Published: March 2005

Using a significantly simplifed modification procedure, four charged analogues of the coenzyme NAD, N(1)‐ and N6‐(2‐hydroxy‐3‐trimethylammoniumpropyl)‐NAD, N(1)‐ and N6‐(3‐sulfopropyl)‐NAD were prepared. The kinetic parameters of these derivatives and N(1)‐(2‐aminoethyl)‐NAD, N6‐(2‐aminoethyl)‐NAD and tricyclic 1,N6‐ethanoadenine‐NAD, all with alterations to the adenine moiety, were determined for porcine heart lactate dehydrogenase isoenzyme H4. The coenzyme activity depends on both position and charge of the introduced groups. Modification of the N6‐position leads to a 25–250‐fold increase of the kcat/Km value compared to the related N(1) derivative. The kcat/Km value for 1,N6‐ethanoadenine‐NAD is in the range between that of N(1)‐(2‐aminoethyl)‐NAD and N6‐(2‐aminoethyl)‐NAD. In the case of both N(1) and N6 functionalization, the Km values increase from (3‐sulfopropyl)‐NAD, with a negatively charged substituent at the adenine, over (2‐aminoethyl)‐NAD to (2‐hydroxy‐3‐trimethylammoniumpropyl)‐NAD with an uncharged and positively charged substituent, respectively, at the adenine. All N6 derivatives are analogues like NAD with respect to Km and/or Vmax and kcat/Km. The conformation of NAD and its derivatives was calculated and their interaction in the active site of lactate dehydrogenase was simulated using the molecular mechanics programm AMBER. The significant differences in activity in correlation to porcine heart lactate dehydrogenase isoenzyme H4 could be rationalized by modelling the three‐dimensional structure of the NAD site.
Functional coupling between sarcoplasmic‐reticulum‐bound creatine kinase and Ca2+‐ATPase
Authors: KORGE Paavo , BYRD Shere K. , CAMPBELL Kenneth B.
Published: March 2005

We investigated the role of creatine kinase bound to sarcoplasmic reticulum membranes of fast skeletal muscle in the local regeneration of ATP and the possible physiological significance of this regeneration for calcium pump function. Our results indicate that ADP produced by sarcoplasmic reticulum Ca2+‐ATPase is effectively phosphorylated by creatine kinase in the presence of creatine phosphate. This phosphorylation is an important function of the membrane‐bound creatine kinase because accumulation of ADP has a depressive effect on Ca2+‐uptake by sarcoplasmic reticulum vesicles. The concentration‐dependent depression of Ca2+‐uptake by ADP was especially pronounced when there was strong back inhibition by high intravesicular [Ca2+]. ATP regenerated by endogenous creatine kinase was not in free equilibrium with the ATP in the surrounding medium, but was used preferentially by Ca2+‐ATPase for Ca2+‐uptake. Efficient translocation of ATP from creatine kinase to Ca2+‐ATPase, despite the presence of an ATP trap in the surrounding medium, can be explained by close localization of creatine kinase and Ca2+‐ATPase on the sarcoplasmic reticulum membranes. These results suggest the existence of functional coupling between creatine kinase and Ca2+‐ATPase on skeletal muscle sarcoplasmic reticulum membranes. Several factors (amount of membrane‐bound creatine kinase, oxidation of SH groups of creatine kinase, decrease in [phosphocreatine]) can influence the ability of creatine kinase/phosphocreatine system to support a low ADP/ATP ratio and fuel the Ca2+‐pump with ATP. These factors may become operative in the living cells, influencing functional coupling between creatine kinase and Ca2+‐ATPase and may have an indirect effect on Ca2+‐pump function before Ca2+‐ATPase itself is affected.
Acetyl‐acyl carrier protein is not a major intermediate in fatty acid biosynthesis in spinach
Authors: JAWORSKI Jan G. , POST‐BEITTENMILLER Dusty , OHLROGGE John B.
Published: March 2005

The extent to which acetyl–acyl carrier protein (acetyl‐ACP) is an intermediate in fatty acid biosynthesis was examined. Acetyl‐ACP was the least effective primer of fatty acid synthesis by spinach extracts when compared to acetyl‐CoA, butyryl‐ACP or hexanoyl‐ACP. Furthermore, the rate of acetyl‐ACP‐primed fatty acid synthesis was inhibited significantly by cerulenin, indicating that the slow utilization of acetyl‐ACP was predominantly by 3‐oxoacyl‐ACP synthase I. In light‐incubated isolated chloroplasts with high rates of fatty acid synthesis (greater than 800 nmol·h−1·mg chlorophyll−1), the rate of acetyl‐ACP metabolism was at least 10–30‐fold slower than the rate of butyryl‐ACP metabolism. The relatively slow metabolism of acetyl‐ACP provided in situ evidence that (a) butyryl‐ACP was formed principally from condensation of malonyl‐ACP with acetyl‐CoA and (b) acetyl‐ACP was a minor participant in fatty acid biosynthesis.
Human 4‐hydroxyphenylpyruvate dioxygenase
Authors: RÜETSCHI Ulla , DELLSÉN Anita , SAHLIN Pelle , STENMAN Göran , RYMO Lars , LINDSTEDT Sven
Published: March 2005

We report the primary structure of 4‐hydroxyphenylpyruvate dioxygenase [4‐hydroxyphenylpyruvate: oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver λgt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino‐acid sequence. These peptide sequences covered 86% of the cDNA‐derived amino‐acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa.

There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4‐hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P. J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C‐terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4‐hydroxyphenylpyruvate dioxygenases. The identity of the C‐terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i. e. amino acids which have been implicated as ligands for iron in 2‐oxoacid‐dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14 → qter by Southern‐blot analysis of human‐rodent somatic‐cell hybrids.
Alcohol esterification reactions and mechanisms of snake venom 5′‐nucleotide phosphodiesterase
Authors: GARCÍA‐DÍAZ Miguel , AVALOS Martín , CAMESELLE José Carlos
Published: March 2005

In a previous study we have shown that snake venom 5′‐nucleotide phosphodiesterase (SVP) catalyzes methanol‐esterification reactions [García‐Díaz, M., Avalos, M. & Cameselle, J. C. (1991) Eur. J. Biochem. 196, 451–457]. Now we have demonstrated that SVP catalyzes AMP transfer from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2‐chloroethanol or 2,2‐dichloroethanol. The AMP‐O‐alkyl ester products were identified by HPLC, enzyme analysis, ultraviolet and NMR spectroscopy. Those results show the potential of SVP as a tool to prepare 5′‐nucleotide esters and agree with the formation of a covalent 5′‐nucleotidyl‐SVP intermediate susceptible to nucleophilic attack by short‐chain (poly)alcohols as acceptors alternative to water. To test the kinetic influence of the solvent nucleophile in SVP mechanisms, initial rates of ATP solvolysis were assayed in different water/alcohol mixtures. Relatively high alcohol concentrations inactivated SVP but lower concentrations gave proportional rates of alcoholysis. An efficiency parameter (EA), defined as the ratio of the mole fraction of AMP‐O‐alkyl ester as a product to that of alcohol as an acceptor in water/alcohol mixtures, made possible the comparison of alcohols and water as AMP acceptors at low concentrations, as it could be reasoned that EA= 1 for water. Rates of hydrolysis (VH) of substrates yielding AMP and different leaving groups were also assayed. The higher EA and VH values correspondended, respectively, to those acceptors and leaving‐group conjugate acids with lower pK and higher polar‐substituent constants (σ*). The results support the occurrence of general acid‐base catalysis in the active center of SVP and the identification of rate‐limiting steps. A model is proposed for the mechanisms of SVP‐catalyzed hydrolysis and alcoholysis which accounts for the influence of the acid‐base properties of alcohols on the kinetic profile of SVP reaction sequences.
Structure of the O‐specific polysaccharide containing pentitol phosphate, isolated from Hafnia alvei strain PCM 1191 lipopolysaccharide
Authors: GAMIAN Andrzej , ROMANOWSKA Elzbieta , DABROWSKI Ursula , DABROWSKI Janusz
Published: March 2005

The O‐specific polyaccharide of the lipopolysaccharide produced by Hafnia alvei strain PCM 1191 was shown by composition and methylation anlyses, periodate oxidation and one‐ and two‐dimensional 1H‐ and 31P‐NMR spectroscopy to be a polymer of a branched hexasaccharide repeating units having the structure:

image

where lAraol =l‐arabitol

The polysaccharide of 1191 strain has teichoic‐acid‐like character. Its peculiar feature is the presence of arabitol phosphate, a component observed for the first time in bacterial lipopolysaccharides.
Metabolic characteristics of rat liver cytosolic 5SRNA
Authors: OGATA Kikuo , KURAHASHI Ayumi , NISHIYAMA Chikage , TERAO Kazuo
Published: March 2005

5SRNA were purifiéd from the cytosol, ribosomes, nucleoli and nucleoplasm of rat liver. The 5′‐terminal and 3′‐terminal sequences of these 5SRNA species were shown to be the same. The time course of changes in specific activities of these 5SRNA in rat liver, 20 min to 16 h after intraperitoneal injection of [14C]orotic acid, were investigated. The specific activity of cytosolic 5SRNA increased almost linearly for 3 h, without a lag, to a high value, then decreased rapidly. The specific activity of cytosolic 5SRNA was much higher than the specific activities of 5SRNA from the other cellular components over 45 min to 6 h after intraperitoneal injection, although at 20 min the specific activity was similar to that of nucleoplasmic 5SRNA. Thus, cytosolic 5SRNA of rat liver is characterized by a high rate of turnover, which was shown to be comparable to that of heterogeneous nuclear RNA. The specific activity of 5SRNA, 45 min to 6 h after intraperitoneal injection, was as follows; cytosolic > nucleoplasmic > nucleolar > ribosomal 5SRNA. The specific activity of nucleoplasmic RNA that migrated slightly slower than nucleoplasmic 5SRNA on PAGE was much higher than that of cytosolic 5SRNA or nucleoplasmic 5SRNA, and so may be the precursor of nucleoplasmic 5SRNA. Together, these results indicate that cytosolic 5SRNA is transferred from the nuclei to the cytosol immediately after maturation of the precursor of nucleoplasmic 5SRNA without entering the nucleoplasmic pool of 5SRNA.
Characterization of the high‐affinity oligosaccharide‐binding site of the 205‐kDa porcine large granular lymphocyte lectin, a member of the leukocyte common antigen family
Authors: BEZOUŠKA Karel , KRAJHANZL Alexandr , POSPÍŠIL Miloslav , KUBRYCHT Jaroslav , STAJNER Karel , FELSBERG Jürgen , KOCOUREK Jan
Published: March 2005

Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non‐major‐histocompatibility complex‐restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [PospÍS̆il M., Kubrycht J., Bezous̆ka K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83–90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205‐kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4‐azidophenyl (photoactivatable) and 4‐hydroxyphenyl (radio‐iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N‐glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high‐affinity ligands to investigate the oligosaccharide‐combining site of the 205‐kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205‐kDa polypeptide constitutes the major cell‐surface cacium‐independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand‐labeled and eleven other peptides proved that the 205‐kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high‐affinity binding site is localized in the N‐terminal 70‐kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate‐recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O‐glycosidically linked lineage‐specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell‐surface glycoproteins expressed high‐affinity binding sites for the oligosaccharide ligands. Thus, N‐linked oligosaccharide moieties of cell‐surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide‐mediated activation of lymphocytes.
Mössbauer spectroscopy of metalloproteins and the use of Fourier transforms
Authors: DUNHAM W. R. , HARDING L. J. , SANDS R. H.
Published: March 2005

A method for obtaining accurate, quantitative 57Fe Mössbauer spectra from biological samples is illustrated stepwise in a data reduction procedure. Exact criteria are presented for deciding when it is necessary to account for the effects of the Beer‐Lambert law in the Mössbauer spectra from biological samples. This procedure makes extensive use of the fast Fourier transform and other computer techniques in its data reduction and its curve‐fitting stages. A method for optimizing sample thickness is presented. The choice of truncation in Fourier space as a means to numerically stabilize the deconvolution procedure is defended. Several advantages for curve fitting in Fourier space are shown. Maximization of information content is discussed for Mossbauer spectral simulation techniques.
Dye‐linked l‐malate dehydrogenase from thermophilic Bacillus species DSM 465
Authors: OHSHIMA Toshihisa , TANAKA Satohiro
Published: March 2005

The distribution of dye‐linked l‐malate dehydrogenase (l‐malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria. The enzyme occurred widely in thermophilic spore‐forming bacteria like bacilli and thermoactinomycetes. The enzyme was purified to homogeneity from a thermophile, Bacillus sp. DSM 465, with a 2.7% overall recovery by DEAE‐Toyopearl column chromatography, Sephacryl S‐400 column chromatography and preparative slab PAGE. The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa. The enzyme retained its full activity upon heating at 55°C for at least 60 min and with incubation at pH 5.0–10.0, 55°C, for 10 min. The enzyme exclusively catalyzed l‐malate dehydrogenation in the presence of an electron acceptor such as 2,6‐dichloroindophenol. The Michaelis constants for l‐malate and 2,6‐dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively. FAD was identified as a prosthetic group of the enzyme by HPLC.
Biochemical bases of the interaction of human basic fibroblast growth factor with glycosaminoglycans
Authors: COLTRINI Daniela , RUSNATI Marco , ZOPPETTI Giorgio , ORESTE Pasqua , ISACCHI Antonella , CACCIA Paolo , BERGONZONI Laura , PRESTA Marco
Published: March 2005

In the present study we have attempted a characterization of the biochemical bases of the interaction of human basic fibroblast growth factor (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfated compounds to protect bFGF and different bFGF mutants from tryptic cleavage.

Heparin protects bFGF from trypsin digestion in a dose‐dependent fashion. Substitution by site‐directed mutagenesis of two or more basic residues with neutral glutamine residues in the aminoterminal region bFGF(27–32) or in the carboxyl‐terminal region bFGF(118–129) does not significantly affect the protective effect exerted by heparin. In contrast, heparin protection is abolished when the full region bFGF(27–32) is deleted.

The capacity of different GAGs to protect bFGF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interaction with bFGF. This is confirmed by the different capacity of various sulfated compounds (including dextran sulfates, suramin, trypan blue, and sulfate ion) to protect bFGF from tryptic digestion. Moreover, tryptic digestion studies performed with various heparin molecules and dextran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indicate that the number of bFGF molecules which interact with a single molecule of polysaccharide is related to the molecular mass of the GAG and that six hexose residues are sufficient to protect 1–2 molecules bFGF.

In conclusion, our findings indicate that the capacity of GAGs to protect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requirements of the bFGF molecule. These studies will help to design synthetic oligosaccharides endowed with different bFGF agonist and/or antagonist activities.
Relationships between alkali light‐chain complement and myosin heavy‐chain isoforms in single fast‐twitch fibers of rat and rabbit
Authors: WADA Masanobu , PETTE Dirk
Published: March 2005

The present study compares the alkali myosin light chain (LC) complement of the fast fiber types IIB, IID and IIA in single fibers from rat muscle, as well as in type IID and type IIA fibers from rabbit muscle. Single fibers were classified according to their electrophoretically determined myosin heavy chain (HC) isoforms, HCIIb, HCIId, and HCIIa. Alkali myosin light chains were analysed by densitometric evaluation of two‐dimensional electrophoresis performed on extracts from the same fibers. On the average, the fraction of LC3f, i.e. LC3f/(LC1f+LC3f), was highest in type IIB fibers and lowest in type IIA fibers. Type IID fibers occupied an intermediate position. Also in the rabbit, type IID fibers displayed a higher fraction of LC3f than type IIA fibers. Large scattering of the LC3f fraction in IIB, IID, and IIA fibers indicated that each fiber type is composed of fibers identical with regard to their specific myosin heavy chain complement, but heterogeneous with regard to their fast alkali light chain composition and the resulting light‐chain‐based isomyosins. It is suggested that the variable proportions of the two alkali light chains in the three fast fiber populations serve as a fine tuning of contractile velocities within the ranges determined by the three fast myosin heavy‐chain isoforms.
Catalytic antibody activity elicited by active immunisation
Authors: GALLACHER Gerard , JACKSON Caroline S. , SEARCEY Mark , GOEL Rajiv , MELLOR Geoffrey W. , SMITH Carmen Z. , BROCKLEHURST Keith
Published: March 2005

The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two‐year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4‐nitrophenyl 4‐carboxyme‐thylphenyl hydrogen phosphate and amino groups of keyhole‐limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18‐week immunisation programme using an analogous sulphone immunogen (comprising the amide conjugate bonded through the amino group of 4‐nitrobenzyl, 4‐(4‐aminobutoxy)benzyl sulphone and carboxyl groups of keyhole limpet haemocyanin) were evaluated kinetically by using 4‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenylcarbonate and 4‐nitrophenyl 4‐(2‐hydroxyethoxy)phenyl carbonate as substrates.

Catalytic activity was found in all 13 samples of anti‐phosphate IgG but was absent in the sample of anti‐sulphone IgG as well as in all samples of IgG isolated from the serum of nonimmunised animals. These findings, taken together with the lack of catalytic activity of the antiphosphate IgG towards the 2‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti‐phosphate IgG preparation is entirely antibody‐mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti‐phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation.

The nature of the, albeit small, variation in the catalytic characteristics of the anti‐phosphate IgG (increase in both kcat, the catalytic rate constant calculated as V/2[IgG] and kcat/Km, the apparent second‐order rate constant for the overall catalysed conversion of substrate to products, with increase in Km suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis.

This interpretation is supported by the difference in the values of the dissociation constant Ki for the competitive inhibition by the transition‐state analogue 4‐methylphenyl 4‐nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti‐phosphate IgG samples: for the catalysis with Km= 4.5 μM, Ki= 9 nM and for that with Km= 1.3 μM, Ki= 80 nM.

4‐Nitrobenzyl 4‐(4‐aminobutoxy)benzyl sulphone, the hapten that was used to prepare the sulphone immunogen mentioned in (1), failed to inhibit the hydrolysis of 4‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenyl carbonate catalysed by anti‐phosphate IgG. The sulphone moiety, therefore, does not appear to mimic adequately the carbonate in either ground‐state or transition‐state complexes.
Modulation of thrombin‐induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol‐protease inhibitory sequence of kininogens and S‐(3‐nitro‐2‐pyridinesulfenyl)‐cysteine
Authors: PURI Rajinder N. , MATSUEDA Rei , UMEYAMA Hideaki , BRADFORD Harlan N. , COLMAN Robert W.
Published: March 2005

Thrombin‐induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High‐molecular‐mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln‐Val‐Val‐Ala‐Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe‐Gln‐Val‐Val‐Cys(Npys)‐Gly‐NH2(Npys, 3‐nitro‐2‐sulfenylpyridine), P1, corresponding to the thiol‐protease‐binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin‐induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin‐induced platelet‐shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin‐induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.
Phosphatase 2A associated with polyomavirus small‐T or middle‐T antigen is an okadaic acid‐sensitive tyrosyl phosphatase
Authors: CAYLA Xavier , BALLMER‐HOFER Kurt , MERLEVEDE Wilfried , GORIS Jozef
Published: March 2005

Papovavirus tumor antigens have been shown to associate with the cellular phosphoserine/threonine‐specific protein phosphatase 2A (PP2A). We were interested in the consequences that T‐antigen association might have on PP2A activity and so studies of the phosphatase activity in immunoprecipitates, prepared from polyoma virus‐transformed or polyoma virus‐infected mouse 3T3 fibroblasts, were performed. The phosphoserine/threonine phosphatase activity, measured with phosphorylase a as the substrate, showed all the characteristics of PP2A. It was stimulated by polycations, inhibited by fluoride or p‐nitrophenyl phosphate, sensitive to okadaic acid and microcystin and insensitive to inhibitor‐1 and inhibitor‐2. Phosphotyrosyl phosphatase (PTPase) activity was associated with the middle‐T/small‐T‐associated complex when reduced, carboxamidomethylated and maleylated lysozyme, phosphorylated exclusively on tyrosyl residues, was used as the substrate. This PTPase activity was as sensitive to okadaic acid as was the phosphorylase phosphatase activity; it could be inhibited by phosphorylase a and did not dephosphorylate poly(Glu80Tyr20). The level of middle‐T/small‐T‐associated PTPase activity relative to the phosphorylase phosphatase activity was tenfold higher than that of the purified dimeric PP2A. A similar activity ratio was observed with the purified phosphatase after stimulation with a cellular protein, designated phosphotyrosyl phosphatase activator. These results suggest that the same enzyme may possess dual specificity. In contrast to the cellular trimeric PP2A, containing the 55‐kDa putative regulatory subunit, the middle‐T/small‐T‐associated enzyme had low activity towards a retinoblastoma peptide phosphorylated by p34ede2. These results indicate how middle‐T/small‐T might effect the activity of PP2A in polyoma virus‐transformed cells.
Purification and photoaffinity labeling of fusicoccin receptors from maize
Authors: ADUCCI Patrizia , BALLIO Alessandro , FOGLIANO Vincenzo , FULLONE Maria Rosaria , MARRA Mauro , PROIETTI Noemi
Published: March 2005

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin‐binding specificity) by using an original multi‐step HPLC procedure. The method, based on a combination of adsorption, ion‐exchange and gel‐filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000‐fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa.

Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90‐kDa band is radiolabeled, whereas, as the irradiation time increases, a 30‐kDa band becomes radiolabeled and less radioactivity is detected in the 90‐kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90‐kDa band. Our data suggest that the 90‐kDa protien is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30‐kDa protein as the true receptor.
Sequential assignment of proton resonances in the NMR spectrum of Zn‐substituted α chains from human hemoglobin.
Authors: MARTINEAU Laure , CRAESCU Constantin T.
Published: March 2005

We constructed an artificial holoprotein as a complex between α globin from human adult hemoglobin and the protoporphyrin IX‐Zn(II). The prosthetic group is bound in a single conformation to the apoglobin via a coordinative bond between Zn(II) ion and the proximal histidine (His87). The complex is diamagnetic and does not bind either CO nor O2 thus representing a diamagnetic model of deoxygenated α chains. In the present paper we report extensive resonance assignment in the proton nuclear magnetic resonance spectrum of the Zn‐substituted α chains in phosphate buffer pH 5.6. A large number of aromatic and aliphatic side chain spin systems were identified in the two‐dimensional homonuclear COSY spectra. Based on the assigned resonances of heme substituent protons and their NOE cross‐peaks, we assigned the majority of resonances representing the heme pocket side chains. Using the main‐chain‐directed assignment strategy, we could establish several continuous patterns of sequential assignment and identify partial or total spin systems for a large number of side chains. The final assignment corresponds to 73% of the amino acids. Analysis of chemical shift of assigned resonances and of nuclear Overhauser enhancement connectivities provides structural information on the global and local tertiary conformation in solution and on the ligand‐induced conformational changes. Comparison of observed and calculated ring current shifts enabled us to compare the solution structure with the X‐ray crystal structure of α subunits in deoxy and carbonmonoxy hemoglobin. The global tertiary structure of unliganded chains is highly similar to both liganded and unliganded counterparts in the crystalline state. On the distal side of the heme pocket, Val62 is significantly closer to the heme center, in agreement with its conformation in the crystallographic structure. In contrast, the position of the proximal histidine (His87) relative to the heme is clearly more closely related to that in the liganded tetramer in the crystalline state. Comparison of the chemical shift values for the resonances in carbon monoxy and Zn(II)‐substituted α chains indicates that the ligand‐induced conformational changes are essentially localized in the heme pocket area and affect proximal side residues more than the distal side ones. Some notable spectral changes are discussed in connection with the crystallographic data and their relevance for the functional mechanism.
Structure of a novel glycopeptidolipid antigen containing a O‐methylated serine isolated from Mycobacterium xenopi
Authors: RIVIÈRE Michel , AUGÉ Sophie , VERCAUTEREN Joseph , WISINGEROVÀ Eva , PUZO Germain
Published: March 2005

GPL X‐1, a novel glycopeptidolipid (GPL) isolated from Mycobacterium xenopi (CIPT 140 35004), has recently been found to typify a new class of mycobacterial glycopeptidolipids devoid of C‐mycoside core structure, the so‐called serine‐containing glycopeptidolipid [Rivière, M. & Puzo, G. (1991) J. Biol. Chem. 266, 9057–9063]. Here we report the purification and characterization of a novel serine‐containing GPL termed GPL X‐IIb, isolated from the M. xenopi strain NCTC 10042. On thin‐layer chromatography, this GPL was found to be present in some other M. xenopi strains isolated from patients with pulmonary infections. The sugar and amino‐acid compositions of this GPL were elucidated from the native form using a combination of two‐dimensional homonuclear and heteronuclear scalar coupling NMR. The peptide and sugar sequences, as well as the methoxyl group locations on the C‐3 of the 6‐decxy‐α‐l‐talopyranoside (6dTalp) and on a Ser, were unambiguously determined by heteronuclear multiple‐bond correlation experiments. GPL X‐IIb was found to be composed of a lipotetrapeptide of the following structure C12‐Ser‐OMe‐Ser‐Phe‐aThr‐OMe (aThr = allothreonine). The sugar part is made up of 3OMe‐α‐l‐6dTalp and the following disaccharide: α‐l‐Rhap‐(1→3)‐2‐O‐Lau‐α‐l‐Rhap (Rhap= rhanmopyranose). Unlike GPL X‐I, the sugar attachment sites on the tetrapeptide were successfully determined from heteronuclear three‐bond coupling correlation observed in the heteronuclear multiple bond correlation spectrum between the anomeric carbon resonances and the β protons of aThr‐OMe and Ser. It was established that the 3OMe‐6dTalp glycosylates the Ser while the disaccharide is linked to the aThr‐OMe. Thus both GPL X‐I and GPL X‐IIb share a common lipotetrapeptide core [with the exception of Ser(OMe)] but drastically differ in their oligosaccharide appendage. Thus, by analogy with the M. avium complex, the present report suggests that M. xenopi species can be divided in various serovars characterized by the unique structure of their C‐mycoside GPL oligosaccharide appendage, enhancing the interest for this new type of serine‐containing glycopeptidolipid.