bluuebunny/crossref_metadata_2025 huggingface dataset:
The stoichiometry of the tightly bound NAD+in urocanase
Authors: KLEPP Jürgen , RÉTEY János
Published: March 2005

Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979)J. Biol. Chem. 254, 843–851] fromPseudomonas putidawas submitted to high‐performance liquid chromatography on a TSK‐DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively.

The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm.

Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK‐DEAE column confirmed their partial transformation into peak A.

Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8–1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7‐14C]nicotinate into urocanase using a nicotinate auxotrophic mutant ofP. putida.

Form A of urocanase showed, after treatment with NaBH4up to 50% inhibition, an elution pattern (TSK‐DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated.

We conclude that form A of the urocanase dimer contains two intact NAD+molecules. In form B one of the two subunits contains an NAD+‐nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+in both subunits. Intact NAD+can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.
Butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat
Authors: SANDOR Attila , HOPPEL Charles L.
Published: March 2005

Urinary excretion of total carnitine in 48‐h fasted rats dropped to 0.30 ± 0.01 μmol/day from 2.23 ± 0.4 μmol/day found in fed, control animals (mean ± SEM). Despite this marked retention, the total carnitine content of the whole body remained constant, about 83 μmol, predicting a slow‐down in biosynthesis.

The conversion of butyrobetaine into carnitine takes place only in the liver in rats. 48 h of starvation caused a decrease in the liver butyrobetaine level from 11.6 ± 1.19 nmol/g to 9.30 ± 1.19 nmol/g, which in whole livers corresponds to a decrease from 138 nmol to 61.3 nmol. The conversion rate of butyrobetaine into carnitine was studied with radiolabelled butyrobetaine. 30 min after injection of [3H]butyrobetaine the carnitine pool in the liver of fasted rats was labelled to about the same extent as that in fed rats, but from a butyrobetaine pool with higher specific radioactivity. Therefore, the conversion rate of butyrobetaine into carnitine was reduced. The newly formed carnitine found in the whole body of fasted rats was estimated to be 59% of controls. We conclude that the biosynthesis of carnitine in fasted rats slows down, for which a decreased availability of butyrobetaine in the liver is responsible.

Urinary excretion of butyrobetaine in the fasted group decreased to 74.1 nmol/day from the 222‐nmol/day control value while the butyrobetaine content of whole body did not significantly decrease (2.85 μmol vs. 3.04 μmol). Urinary excretion of trimethyllysine was also depressed.
Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins
Authors: HATAKEYAMA Tomomitsu , KAUFMANN Franziska , SCHROETER Barbara , HATAKEYAMA Tamiko
Published: March 2005

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.
The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for β2‐microglobulin in rat brain
Authors: COLE Timothy , DICKSON Phillip W. , ESNARD Frederic , AVERILL Sharon , RISBRIDGER Gail P. , GAUTHIER Francis , SCHREIBER Gerhard
Published: March 2005

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and β2‐microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma.

The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N‐linked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine.

Rat β2‐microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human β2‐microglobulin cDNA. Tissue levels of β2‐microglobulin mRNA in the rat were measured by hybridization to rat β2‐microglobulin cDNA. The highest levels of β2‐microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of β2‐microglobulin mRNA.
Purification and characterization of a protease from Xenopus embryos
Authors: MIYATA Shohei , YOSHIDA Yo‐ichi , KIHARA Hirozi K.
Published: March 2005

A proteolytic enzyme was purified from Xenopus embryos. The purification procedure consisted of fractionation of an extract of embryos with acetone, gel filtration of Sephadex G‐75 and chromatography on carboxymethyl‐cellulose and hydroxylapatite. The preparation of enzyme appeared to be homogeneous as judged by electrophoresis in polyacrylamide gels. This protease had a molecular mass of 43–44 kDa and was composed of two subunits with molecular masses of 30 kDa and 13 kDa. The optimal pH of the reaction catalysed by the protease was approximately 4.0. This proteolytic activity was inhibited by antipain, leupeptin and iodoacetic acid; it was not affected by phenylmethylsulfonyl fluoride and pepstatin; and it was enhanced by dithiothreitol. In the presence of RNA, the optimal pH was shifted from pH 4.0 to pH 4.5. The protease was activated by addition of total RNA from Xenopus embryos, by poly(rU) or poly(rG). In contrast, after addition of tRNA or poly(rC), no activation of the protease was observed.
Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP
Authors: BAROUKI Robert , PAVÉ‐PREUX Muriel , BOUSQUET‐LEMERCIER Bernadette , POL Stanislas , BOUGUET Jacqueline , HANOUNE Jacques
Published: March 2005

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6–10‐fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 μM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 μM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8‐bromoadenosine 3′,5′‐monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2–3‐fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8‐cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments.

Northern blot analysis revealed the presence of two major (2.1‐kb and 1.8‐kb) and one minor (4‐kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.
The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNAfMet
Authors: PERREAULT Jean‐Pierre , PON Richard T. , JIANG Mei‐yan , USMAN Nassim , PIKA Jana , OGILVIE Kelvin K. , CEDERGREN Robert
Published: March 2005

Stepwise, solid‐phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNAfMet. The 34‐ribonucleotide oligomer corresponding to the sequence of the 5′‐half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34‐nucleotide oligomer serves as an acceptor in the RNA‐ligase‐catalyzed reaction with a phosphorylated 43‐ribonucleotide oligomer corresponding to the sequence of the 3′‐half molecule of tRNAfMet. The DNA molecule having the sequence of tRNAfMet is a 76‐deoxyribonucleotide oligomer with a 3′‐terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2′‐hydroxyl groups except for the 3′‐terminal 2′‐OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNAfMet. The all‐RNA 77‐nucleotide oligomer can be aminoacylated by E. coli methionyl‐tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNAPhe and tDNALys, the rA77‐DNAfMet can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNAfMet does not depend on the presence of 2′‐hydroxyl groups with the exception of that in the 3′‐terminal nucleotide.
A theoretical treatment of damped oscillations in the transient state kinetics of single‐enzyme reactions
Authors: RYDE‐PETTERSSON Ulf
Published: March 2005

An extension of the available kinetic theory for reactions in the transient state is presented which establishes that single‐enzyme reactions may exhibit damped oscillations under the conditions of standard kinetic experiments performed by stopped‐flow techniques. Such oscillations may occur for reasonable magnitudes of rate constants in the enzymic reaction mechanism and at physiological concentrations of enzyme and substrate. In the simplest reaction systems, the oscillations will be strongly damped and lead to progress curves resembling those of a reaction governed by standard exponential transients; statistical regression methods may then have to be applied for their detection and characterization. The observation that single‐enzyme reactions may exhibit oscillatory behaviour points to a previously unrecognized possible source of the damped oscillations observed in metabolic systems such as the pathways of glycolysis or photosynthesis.
Purification, characterization and immunological properties of 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase from maize (Zea mays) seeds
Authors: GRAÑA Xavier , UREÑA Jesús , LUDEVID Dolores , CARRERAS José , CLIMENT Fernando
Published: March 2005

2,3‐Bisphosphoglycerate‐independent phosphoglycerate mutase (EC 5.4.2.1) was purified and characterized from maize. SDS electrophoresis showed only one band with a molecular mass of 64 kDa, similar to that determined for the native enzyme by gel‐filtration chromatography. The kinetic constants were similar to those reported for wheat germ phosphoglycerate mutase. Rabbit antiserum against maize phosphoglycerate mutase possesses a high degree of specificity. It also reacts with the wheat germ enzyme but fails to react with other cofactor‐independent or cofactor‐dependent phosphoglycerate mutases. Cell‐free synthesis experiments indicate that phosphoglycerate mutase from maize is not post‐translationally modified.
Secondary structure of 7SK and 7‐2 small RNAs
Authors: SUH Dick , YUAN Yan , HENNING Dale , REDDY Ram
Published: March 2005

Pseudogenes having homology to small RNAs, like 7SL, 7SK, 6S, 4.5S, U1, U2, and U3 RNAs, are abundant and dispersed in the genomes of higher eukaryotes [reviewed in Weiner et al. (1986) Annu. Rev. Biochem. 55, 631–661]. To understand better the possible origin of these pseudogenes, we studied the abilities of cytoplasmic 7SL, 7SK, and nucleolar 7‐2 RNAs to self‐prime and result in the synthesis of cDNAs. When rat 7SK RNA was used as substrate, a 294‐nucleotide‐long cDNA was synthesized in vitro by reverse transcriptase, indicating that the 3′ end of 7SK RNA can act in a self‐priming manner to generate 7SK cDNA. When 7‐2 RNA was used as a substrate, a cDNA of approximately 235 nucleotides was observed; 7SL RNA did not act as a self‐primer.

Earlier studies have shown that DNAs homologous to 7SK RNA are represented by a moderately reiterated family in the mammalian genomes and many of these sequences were found to be truncated 7SK pseudogenes [Murphy et al. (1984) J. Mol. Biol. 177, 575–590]. In this study, one 7SK clone from the rat genome was characterized by sequencing. This clone contained 243 base pairs homologous to the 5′ end of 7SK RNA, and was flanked by direct repeats. These data suggest that, as previously proposed for some U3 pseudogenes [Bernstein et al. (1983) Cell 32, 461–472], one mechanism for the generation of truncated 7SK pseudogenes may be the integration of self‐primed reverse transcripts of 7SK RNA at random genomic sites.
Expression and processing of cyanobacterial Mn‐stabilizing protein in Escherichia coli
Authors: KUWABARA Tomohiko , NAGATA Ryuichi , SHINOHARA Kenji
Published: March 2005

The wox A gene of cyanobacterium Anacystis nidulans R2, which encodes the precursor of the Mn‐stabilizing protein involved in photosynthetic water oxidation, was found to be expressed in Escherichia coli. The 30‐kDa expression product was indistinguishable from the authentic mature protein on SDS/PAGE. Upon fractionation of E. coli cells, the expression product was co‐precipitated with the membrane fraction, which is consistent with the water‐insoluble nature of the authentic mature protein. Analysis of the amino‐terminal amino acid sequence of the product revealed that it is identical to the sequence from the 28th residue of the precursor, indicating that the precursor is processed in E. coli. The expression product was digested by trypsinization of E. coli spheroplasts, but not by that of intact cells. This observation suggests that the product is secreted from the cytoplasmic membrane, but not from the outer membrane. The occurrence of both processing and secretion suggests that a signal peptidase of E. coli can recognize the structure for translocation across the thylakoid membrane. Comparison of the signal sequence and the presequence of sweet potato sporamin A suggests that the processing enzymes of the thylakoid membrane and endoplasmic reticulum possess a common substrate specificity.
Resonance Raman study on the structure of the active sites of microsomal cytochrome P‐450 isozymes LM2 and LM4
Authors: HILDEBRANDT Peter , GREINERT Rüdiger , STIER Anton , TANIGUCHI Hisaaki
Published: March 2005

The isozymes 2 and 4 of rabbit microsomal cytochrome P‐450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100–1700 cm−1 is presented for different ferric states of cytochrome P‐450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme—protein interactions. While in cytochrome P‐450 LM2 the six‐coordinated low‐spin configuration is predominantly occupied, in the isozyme LM4 the five‐coordinated high‐spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low‐spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high‐spin form of the isozyme LM4.

The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type‐I substrate binding. While in cytochrome P‐450 LM4 the occupation of the substrate‐binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground‐state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accomodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high‐spin states in the substrate‐bound and substrate‐free enzyme are structrually different. The formation of the inactive form, P‐420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P‐450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P‐450 isozymes.
The voltage‐dependent activity of Escherichia coli porins in different planar bilayer reconstitutions
Authors: LAKEY Jeremy H. , PATTUS Franc
Published: March 2005

Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt‐extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl‐polyoxyethylene (Octyl‐POE). These methods represent the major approaches to porin isolation and purification. All three were reconstituted into Schindler‐type bilayers. Detergent‐solubilised OmpF was also reconstituted into Montal‐Mueller‐ and Mueller‐Rudin‐type bilayers. In all cases voltage‐dependent closing of OmpF was observed. Octyl‐POE‐extracted PhoE porin was similarly investigated in all three types of planar bilayer. Two membrane‐formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained. Firstly, porins in membranes formed by the Montal‐Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation. Secondly, membranes formed by the Mueller‐Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity.
Dependence on membrane components of methanogenesis from methyl‐CoM with formaldehyde or molecular hydrogen as electron donors
Authors: DEPPENMEIER Uwe , BLAUT Michael , GOTTSCHALK Gerhard
Published: March 2005

Methane formation from 2‐(methylthio)‐ethanesulfonate (methyl‐CoM) and H2 by the soluble fraction from the methanogenic bacterium strain Gö1 was stimulated up to tenfold by the addition of the membrane fraction. This stimulation was observed with membranes from various methanogenic species belonging to different phylogenetic families, but not with membranes from Escherichia coli or Acetobacterium woodii. Treatment of the membranes with strong oxidants, i.e. O2 and K3[Fe(CN)6], or with SH reagents, i.e. Ag+, p‐chloromercuribenzoate or iodoacetamide, caused an irreversible decrease or loss in stimulatory activity, as did heat treatment at temperatures above 78°C. Methanogenesis from methyl‐CoM with formaldehyde instead of H2 as electron donor depended similarly on the membrane fraction. With membranes, 1 mol HCHO was oxidized to 1 mol CO2 and allowed the formation of 2 mol CH4 from 2 mol CH3‐CoM. Without membranes, per mol of HCHO oxidized 1 mol H2 was formed and 1 mol CH4 was produced from CH3‐CoM; the rate was 10–20% of that in the presence of membranes. When methyl‐CoM was replaced by an artificial electron acceptor system consisting of methylviologen and metronidazole, the formaldehyde‐oxidizing activity was no longer stimulated by the membrane fraction. These results demonstrate for the first time an essential function of membrane components in methanogenic electron transfer.
Coenzyme specificity of mammalian liver d‐glycerate dehydrogenase
Authors: Van SCHAFTINGEN Emile , DRAYE Jean‐Pierre , Van HOOF François
Published: March 2005

d‐Glycerate dehydrogenase (glyoxylate reductase) was partially purified from rat liver by anion‐ and cation‐exchange chromatography. When assayed in the direction of d‐glycerate or glycolate formation, the enzyme was inhibited by high (≥0.5 mM), unphysiological concentrations of hydroxypyruvate or glyoxylate much more potently in the presence of NADPH than in the presence of NADH. However, the dehydrogenase displayed a much greater affinity for NADPH (Km < 1 μM) than for NADH (Km= 48–153 μM). Furthermore, NADP was over 1000‐fold more potent than NAD in inhibiting the enzyme competitively with respect to NADH. NADP also inhibited the reaction competitively with respect to NADPH whereas NAD, at concentrations of up to 10 mM had no inhibitory effect. When measured by the formation of hydroxypyruvate from d‐glycerate, the enzyme also displayed a much greater affinity for NADP than for NAD. These properties indicate that liver d‐glycerate dehydrogenase functions physiologically as an NADPH‐specific reductase.

In agreement with this conclusion, the addition of hydroxypyruvate or glyoxylate to suspensions of rat hepatocytes stimulated the pentose‐phosphate pathway. The coenzyme specificity of d‐glycerate dehydrogenase is discussed in relation to the biochemical findings made in d‐glyceric aciduria and in primary hyperoxaluria type II (l‐glyceric aciduria).
Identification of P2Y purinoceptors associated with voltage‐activated cation channels in cardiac ventricular myocytes of the rat
Authors: BJÖRNSSON Ólafur G. , MONCK Jonathan R. , WILLIAMSON John R.
Published: March 2005

Extracellular ATP has vasodilatory and inotropic effects in the heart. We have demonstrated that extracellular ATP, in a concentration‐dependent manner (10 nM–0.1 mM), increased [Ca2+]i in suspensions of isolated fura‐2‐loaded rat cardiac ventricular myocytes (maximum 96 ± 10% increase over basal levels, SEM, n= 12, P < 0.01). The increase in [Ca2+]i was often biphasic, with an initial fast phase (< 1s) of low amplitude, followed by a slower phase of higher amplitude. A second application of ATP had little effect, and ATP abolished the effect of subsequent electrical stimulations, even through the cells were still able to respond with an increase in [Ca2+]i to KCl‐induced depolarization or stimulation by caffeine. Pretreatment of cells with nifedipine, verapamil, caffeine, ryanodine, or 8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxybenzoate hydrochloride attenuated the effect of extracellular ATP on [Ca2+]i, and binding of extracellular free calcium by excess EGTA completely abolished the effects of extracellular ATP and electrical stimulation. Extracellular ATP increased bisoxonol fluorescence in ventricular myocytes, indicating depolarization of the sarcolemma. Pretreatment of the myocytes with tetrodotoxin (50 μM), or replacement of NaCl in the incubation buffer with the impermeant cation N‐methyl‐D‐glucamine, suppressed the extracellular ATP effect on [Ca2+]i. ADP and AMP had smaller effects on [Ca2+]i than ATP; adenosine had no effect. ATP analogues showed the following rank order of potency in increasing [Ca2+]i or bisoxonol fluorescence: ATP ≥ 2‐methylthioATP ∼ adenosine 5′‐O‐[3‐thio]triphosphate > adenosine 5′‐[α,β‐methylene]triphosphate Gt; adenosine 5′‐[β,γ‐methylene]triphosphate ≫ adenosine 5′‐[β,γ‐imino]triphosphate > adenosine. These data are consistent with the presence of purinoceptors (P2Y subtype) on the sarcolemma of cardiac ventricular myocytes of the rat, which upon activation lead to depolarization and activation of cation channels of the sarcolemma and flux of extracellular Ca2+ into the cells. This may result in further flux of Ca2+ into the cytosol from intracellular stores. The effects of extracellular ATP on [Ca2+]i in rat cardiac ventricular myocytes may, in part, explain the direct inotropic effects of extracellular ATP on the mammalian heart.
Extracellular ATP stimulates poly(inositol phospholipid) hydrolysis and eicosanoid synthesis in mouse peritoneal macrophages in culture
Authors: PFEILSCHIFTER Josef , THÜRING Barbara , FESTA Fiorentina
Published: March 2005

The effects of extracellular ATP on inositol phospholipid breakdown and synthesis of eicosanoids were studied in mouse peritoneal macrophages. Addition of ATP to intact cells labelled with [3H]inositol stimulated a rapid (within 10 s) formation of inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate. In parallel there was also a substantial accumulation of inositol 1,3,4‐trisphosphate and the monophosphate and bisphosphate derivatives of inositol. Within 10 s after the addition of 30 μM ATP there was a twofold increase in inositol trisphosphate (InsP3), which declined over 2 min. The ED50 for ATP‐stimulated generation of InsP3 was approximately 12 μM. ADP and GTP showed only weak effects on InsP3 formation, while AMP and adenosine were completely ineffective at 30 μM. Furthermore, the rank order of potency of ATP analogues was ATP > ATP[S] > AdoPP[NH]P= AdoPP[CH2]P > AdoP[CH2]PP thus, indicating the presence of a P2y‐purinergic receptor. Cells labelled with [3H]arachidonic acid showed a 50% increase of label in 1,2‐diacylglycerol after 15 s upon stimulation with ATP. In parallel to the stimulation of inositol phospholipid hydrolysis, ATP also caused a marked synthesis of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in mouse peritoneal macrophages. The rank order of potency of ATP analogues was identical with that of InsP3 generation. The effect on eicosanoid synthesis could be mimicked by the calcium ionophore A23187 and the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate. These results suggest that ATP‐induced activation of P2y‐purinergic receptors in mouse peritoneal macrophages triggers inositol phospholipid breakdown and eicosanoid synthesis.
Purification of active human plasminogen activator inhibitor 1 from Escherichia coli
Authors: LAWRENCE Daniel , STRANDBERG Leif , GRUNDSTRÖM Thomas , NY Tor
Published: March 2005

Plasminogen activator inhibitor 1 (PAI‐1) inhibits both tissue‐type plasminogen activator (tPA) and urokinasetype plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procarvotic expression systems for PAI‐1 and characterized the recombinant glycosylated and non‐glycosylated products, together with a non‐recombinant natural control, produced in the histösarcoma cell line HT 1080. For eucaryotic expression, the PAI‐1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI‐1 protein into an inducible expression vector. Recombinant PAI‐1 from CHO cells was purified approximately 50‐fold in two steps and was indistinguishable from natural PAI‐1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI‐1, from which it was purified approximately 30‐fold, with yields of between 15% and 20%. This PAI‐1 formed 1:1 complexes with uPA and also with the single‐ and two‐chain forms of tPA. Kinetic analysis demonstrated that the procaryoteproduced PAI‐1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI‐1 with association rate constants of approximately 107 M−1 s−1. In contrast to PAI‐1 from eucaryotic cells, the PAI‐1 from E. coli had an inherent activity equal to that of guanidine/HCl‐activated natural PAI‐1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI‐1 was absent. However, at 37°C the procaryote‐produced PAI‐1 lost activity at the same rate as natural PAI‐1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli‐derived PAI‐1, added to human plasma and fractionated by Sephacryl S‐200 chromatography, eluted in two peaks that were similar to those obtained with guanidine‐activated PAI‐1 from eucaryotic cells, suggesting that it bound to the PAI‐1‐binding protein (vitronectin).
Arginyl‐tRNA synthetase from yeast
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich
Published: March 2005

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNAArg‐C‐C‐A by arginyl‐tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D= 800–8500), showing that arginine is 800–8500 times more often incorporated into tRNAArg‐C‐C‐A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10000 and 60000.

In aminoacylation of tRNAArg‐C‐C‐A(3′NH2) discrimination factors D1 are in the range 10–600. From these values and AMP formation stoichiometry, pretransfer proof‐reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNAArg‐C‐C‐A, posttransfer proof‐reading factors II2 could be calculated. II1 values between 2 and 120 show that pretransfer proof‐reading is the main correction step, posttransfer proof‐reading (II2∼ 1–10) plays a marginal role.

Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof‐reading factors. According to a two‐step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical ‘stopper’ model of the amino acid recognition site is discussed.
Primary structure of horse serotransferrin glycans
Authors: CODDEVILLE Bernadette , STRATIL Antonin , WIERUSZESKI Jean‐Michel , STRECKER Gérard , MONTREUIL Jean , SPIK Geneviève
Published: March 2005

Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400‐MHz 1H‐NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b variant are, like in human serotransferrin, of the N‐acetyllactosaminic biantennary type, fully sialylated by two residues of N‐acetylneuraminic acid (Neu5Ac; glycan type I).

In contrast, in addition to this structure, two N‐acetyllactosaminic biantennary isomeric structures named type II‐A and type II‐B sialylated by one Neu5Ac residue and one N‐acetyl‐4‐O‐acetylneuraminic acid [Neu(4,5)Ac2] residue located either at Gal6 or 6′ and one N‐acetyllactosaminic biantennary structure (named type III) sialylated by two residues of Neu(4,5)Ac2, were identified in variants Tf 2a and Tf 4b.

These results demonstrate that in an homozygous preparation of horse serotransferrin Tf 0, the heterogeneity is dependent, on the one hand, on the nature of the neuraminic acid substituting a N‐acetyllactosaminic biantennary structure and, on the other hand, on the number of glycans bound to the polypeptide chain.

Moreover, the differences which exist in the molecular mass of 77.5 kDa, 80 kDa and 82 kDa for serotransferrin variants Tf 2a, Tf 4b and Tf 5b, respectively, are not completely explained by the structure and the number of the glycans suggesting that the three variants should also differ in their polypeptide chain.
Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata
Authors: STUPPERICH Erhard , EISINGER Hans Jürgen , KRäTLER Bernhard
Published: March 2005

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure {Coα, Coβ‐(monocyano, monoaquo)‐phenolyl cobamide} has been deduced from 1H NMR, fast‐atom‐bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p‐cresolyl cobamide {Coα, Coβ‐(monocyano, monoaquo)‐p‐cresolyl cobamide}, which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12{Coα‐[α‐(5,6‐dimethylbenzimidazolyl)]‐Coβ‐cyanocobamide}. Sporomusa synthesized coenzymes of phenolyl cobamide andp‐cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.
Extended X‐ray absorption fine structure (EXAFS) investigations of model compounds for zinc enzymes
Authors: EGGERS‐BORKENSTEIN Peter , PRIGGEMEYER Stefan , KREBS Bernt , HENKEL Gerald , SIMONIS Ursula , PETTIFER Robert F. , NOLTING Hans‐Friedrich , HERMES Christoph
Published: March 2005

A test of the ability of extended X‐ray absorption fine structure (EXAFS) to determine structural information with specific reference to zinc sites in enzymes has been made. X‐ray absorption spectra of 18 compounds of zinc have been measured and the nearest‐neighbour scattering has been interpreted using a Fourier transform and an ab initio technique. Empirical Zn‐N, Zn‐O, Zn‐S and Zn‐Cl amplitude and phase functions have been extracted from Zn(C3H4N2)4(ClO4)2, ZnO, Zn(S2COC2H5)2 and [N(CH3)4]2[ZnCl4], respectively and tabulated as a function of the wavevector with respect to 9660.0 eV X‐ray energy. These amplitude and phase functions were then tested with respect to the other 14 compounds. For a single species of atoms in the first coordination shell the interatomic distances can be established to ± 0.5 pm (± 5 × 10−3Å) whilst when mixed shells exist errors in distances are ±4 pm (± 40 × 10−3Å). Coordination numbers are given to ±16% for the single species case and ±25% for the mixed coordination case. Using the theoretical amplitude and phase functions of McKale et al. [(1988) J. Am. Chem. Soc. 110, 3763–3768] the deduced distances are systematically too small by an average of 0.6 pm (6 × 10−3Å). The errors in the coordination numbers are 18%.
Biochemical characterization of phospholipase D activity from human neutrophils
Authors: BALSINDE Jesús , DIEZ Emilio , FERNANDEZ Belén , MOLLINEDO Faustino
Published: March 2005

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol‐1‐phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2+ ‐independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350–400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was dedected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal‐transducing processes during cell stimulation in human phagocytes.
Identification of a new heparin‐binding protein localized within chick basement membranes
Authors: VIGNY Marc , RAULAIS Daniel , PUZENAT Nathalie , DUPREZ Delphine , HARTMANN Marie Paule , JEANNY Jean Claude , COURTOIS Yves
Published: March 2005

A protein with a molecular mass of 19 kDa has been purified to homogeneity from 11‐day‐old chick embryos using a procedure involving chromatography on heparin–Sepharose, immunoaffinity resin and C4 reversed‐phase. Indirect immunofluorescence studies, using polyclonal and monoclonal antibodies raised against this protein, indicate that it is essentially localized within the basement membranes in early embryonic tissues. After the 18th day of embryonic life and in post‐hatched chicken, this protein could only be detected in some eye basement membranes. It appears to be bound to heparan sulfate chains of the proteoglycan present in these structures. Thus, the protein exhibits similar properties to those previously described for fibroblast growth factors (FGF). such as heparin affinity, molecular mass and localization in the basement membranes. In contrast, this protein is present in much larger amounts than FGFs, at least in 11‐day‐old embryos. Furthermore, the first 17 amino acid residues of the N‐terminal sequence show that it does not strictly correspond to any previously described protein.
Large intramolecular movement in human complement protein C3 induced by methylamine
Authors: ÖSTERBERG Ragnar , NILSSON Ulf , STIGBRAND Torgny , KJEMS Jörgen
Published: March 2005

The reaction of methylamine with complement protein C3, which involves cleavage of a labile thiol ester bond, yields a large intramolecular rearrangement. This is shown by small‐angle neutron and X‐ray scattering using a Fab antibody as a marker. For the C3(Fab) 1:1 complex, the methylamine reaction yields an increase in the radius of gyration, R, from 4.6 nm to 6.0 nm. In the absence of Fab the corresponding R values increase from 4.4 nm to 5.1 nm. It is estimated that the methylamine‐induced increase in R may correspond to a movement of the epitope to a position 5 nm away from the centre of gravity of the C3 molecule. In agreement with this finding, the maximum distance within the C3(Fab) complex increases from 16 nm to 22 nm as a result of the methylamine reaction. In order to explain this conformational change, it is tentatively suggested that the methylamine‐induced cleavage of the C3 thiol ester bond leads to a domain rotation within the C3 molecule. In agreement with this idea, the data is consistent with a model which enables a globular domain within the molecule to rotate without redistributing the molecular mass more than that corresponding to the radii of gyration observed.
Carbamoyl phosphate biosynthesis and partition in pyrimidine and arginine pathways of Escherichia coli
Authors: ROBIN Jean‐Pierre , PENVERNE Bernadette , HERVÉ Guy
Published: March 2005

A procedure for the permeabilization of Escherichia coli cells was adapted to the in situ determination of the catalytic and regulatory properties of the enzymes responsible for the biosynthesis of carbamoyl phosphate and its utilization in the pyrimidine and arginine pathways. Differences in enzyme sensitivity to effectors and changes in pH dependence were observed. Partition of carbamoyl phosphate in the two metabolic pathways could be measured under conditions of substrate saturation. The results obtained will allow to test experimentally the theoretical predictions made by A. Goldbeter (1973) PhD thesis, Université Libre de Bruxelles, on the distribution of carbamoyl phosphate and the oscillation of its intracellular concentration.
Purification and characterization of 55‐kDa protein with 3,5,3′‐triiodo‐l‐thyronine‐binding activity and protein disulfide‐isomerase activity from beef liver membrane
Authors: HORIUCHI Rynya , YAMAUCHI Kiyoshi , HAYASHI Hiroaki , KOYA Sakuji , TAKEUCHI Yoshio , KATO Koichi , KOBAYASHI Masiyuki , TAKIKAWA Hiroo
Published: March 2005

Beef liver membranes were shown to have different kinds of 3,5,3′‐triiodo‐l‐thyronine binding proteins including the 55‐kDa protein which had been reported to have this activity in many cells by affinity labelling with N‐bromoacetyl‐3,5,3′‐[125I]triiodo‐l‐thyronine. In order to characterize the molecular features of these binding proteins, the 55‐kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite. and Mono Q anion‐exchange columns. The purity was confirmed with reversed‐phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55‐kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine‐binding activity with a Kd of 57 nM which is similar to the high‐affinity binding site of the membranes.

The anti‐(55‐kDa protein) sera specifically recognized the 55‐kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti‐(55‐kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide‐isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi‐functional 55‐kDa protein which has triiodothyronine‐binding activity and the activity of protein disulfide‐isomerase, which is also reported to be the β subunit of prolyl‐4‐hydroxylase, glycosylation‐site‐binding protein of oligosaccharyl transferase and iodothyronine 5′‐monodeionidase, could be significant in the action of triiodothyronine towards the target cells.
Structure and activity of proteins from pathogenic fungiPhytophthoraeliciting necrosis and acquired resistance in tobacco
Authors: RICCI Pierre , BONNET Philippe , HUET Jean‐Claude , SALLANTIN Marc , BEAUVAIS‐CANTE Françoise , BRUNETEAU Maud , BILLARD Valérie , MICHEL Georges , PERNOLLET Jean‐Claude
Published: March 2005

The phytopathogenic fungiPhytophthora cryptogeaandPhytophthora capsicicause systemic leaf necrosis on their non‐host tobacco; in culture they release proteins, called cryptogein and capsicein, which elicit similar necrosis. In addition, both proteins protect tobacco against invasion by the pathogenPhytophthora nicotianae, the agent of the tobacco black shank, that is unable to produce such an elicitor. Cryptogein causes visible leaf necrosis starting at about 1 μg/plant, whereas 50‐fold as much capsicein is required for the same reaction. Capsicein induces protection even in near absence of leaf necrosis. The activities of both elicitors are eliminated upon pronase digestion. They are proteins of similarMr(respectively 10323 and 10155) and their complete amino acid sequences were determined. They consist of 98 residues, with some internal repetitions of hexapeptides and heptapeptides. 85% identity was observed between both sequences: only two short terminal regions are heterologous, while the central core is entirely conserved. Secondary structure predictions, hydropathy and flexibility profiles differ only around position 15 and at the C‐terminus; these modifications could play a role in the modulation of their biological activities. After a search of the sequence data bases, they appear to be novel proteins.
Unesterified long‐chain fatty acids inhibit thyroid hormone binding to the nuclear receptor
Authors: INOUE Akira , YAMAMOTO Naoki , MORISAWA Yuji , UCHIMOTO Toshiko , YUKIOKA Munehiko , MORISAWA Seiji
Published: March 2005

Unesterified long‐chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 μM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3‐receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T3‐binding sites, thereby reducing the affinity of the receptor for T3. The affinity of the receptor for oleic acid (Ki) was 1.0 μM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3‐binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3‐binding sites in cells in culture.