bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves
Authors: CHEN Ridong , LE MARÉCHAL Pierre , VIDAL Jean , JACQUOT Jean‐Pierre , GADAL Pierre
Published: March 2005

The cytosolic isocitrate dehydrogenases (NADP‐IDH) were purified to homogeneity from pea roots and green leaves with a high yield by ammonium sulfate precipitation, DEAE‐cellulose chromatography, Sephacryl S‐200 gel filtration, Matrex red‐A affinity chromatography and phenyl‐Superose HR 5/5 HPLC.

Both isoenzymes were dimeric proteins, consisting of two apparently identical 41‐kDa subunits, having similar secondary structures with an α‐helical content of 20% and a β‐pleated sheet content of 43%. Similarly immunoassays suggested that the two isoenzymes were closely related in terms of antigenic determinants. However, the two proteins were distinguishable by their electrophoretic mobilities and amino acid compositions.

The profiles of the two isoenzymes as a function of pH were similar and exhibited a broad pH optimum from 8.5 to 9.0 with Mg2+ as cofactor and 8.0 to 8.5 when Mn2+ was used. Compared to the root isoenzyme, the leaf NADP‐IDH appeared to be more heat‐labile. However, these isoenzymes exhibited similar behavior for thermal denaturation in the presence of bovine serum albumin and were stabilized upon addition of substrate, metal and coenzyme. Their values of activation energy were estimated as 47 kJ/mol. When using Mg2+ as cofactor, the two isoenzymes displayed identical KMn2+m, KDL‐isocitratem and KNADPm values, which were calculated to be 2.1 μM, 5.7 μM and 2.7 μM respectively. With Mg2+ as cofactor, their KMg2+mKDL‐isocitratem and KNADPm values were also not statistically different, being 34.0 μM, 15.2 μM and 2.6 μM for the root NADP‐IDH, and 29.0 μM, 20.3 μM and 3.1 μM for the leaf isoenzyme.

From the above data it can be concluded that although the cytosolic NADP‐IDH in pea roots and leaves are organ‐specific isozymes, their similar physicochemical and kinetic properties suggest that the two isozymes might be involved in identical metabolic functions.
Enhancing effect of a phorbol ester and of retinoic acid on glucocorticoid induction of chenodeoxycholate hydroxylation in hepatoma cultures
Authors: LAMBIOTTE Maurice , CRESTEIL Thierry , THIERRY Nicole
Published: March 2005

In cultures of the differentiated clones Faza 967, Fao and HF1‐4, derived from Reuber hepatoma, physiological doses of glucocorticoid induce chenodeoxycholate 6β‐hydroxylation, a microsomal cytochrome‐P‐450‐mediated activity (enhanced in liver by phenobarbital and not by benzo[α]anthracene). Whereas 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) alone has no effect, the tumor promoter, when added to dexamethasone, enhances this induction. This enhancement, half‐maximum with 10 ng/ml TPA, is a function of the dose between 1 ng/ml and 50 ng/ml; 50 ng/ml (80 nM) increase 4–7‐fold the induction rate (as measured in cultures by the amount of bile acid hydroxylated per 106 cells in 24 h, and in homogenates from treated cells) and 2.5–fold the maximum activity attained by the third day of induction. When added to cultures of the dedifferentiated clone H5, treated with benzo[α]anthracene, TPA does not influence benzo[α]pyrene hydroxylase induction, as shown by the total and relative amounts of the various hydrosoluble benzo[α]pyrene metabolites. TPA does not affect tyrosine aminotransferase induction in dexamethasone‐treated Fao cultures. The enhancement is not suppressed by indomethacin, an inhibitor of prostaglandin synthesis. After dexamethasone removal from induced Faza 967 cultures, addition of TPA to the medium does not affect the decay rate of the chenodeoxycholate‐hydroxylating activity.

Retinoic acid similarly enhances the induction by dexamethasone of chenodeoxycholate hydroxylation, both in treated Faza 967 cultures and in homogenates from treated cultures. The effects of TPA and retinoic acid are additive.

These results suggest a possible cooperation at the transcriptional level between transactive factors, involving TPA‐mediated alterations, retinoic acid and glucocorticoid receptors. The system described might provide a convenient experimental approach in the study of its mechanism.
The role of modifications in oligonucleotides in sequence recognition by MvaI restriction endonuclease
Authors: KUBAREVA Elena A. , PEIN Claus‐Dietmar , GROMOVA Elisaveta S. , KUZNEZOVA Svetlana A. , TASHLITZKI Vadim N. , CECH Dieter , SHABAROVA Zoe A.
Published: March 2005

The interaction of MvaI restriction endonuclease with 14‐membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m5dC for the internal dC residue, as well as substitution of fl5dU or rU for dT did not influence the initial rate of hydrolysis (vo) of modified strands, whereas the hydrolysis of unmodified strands was inhibited in some cases. Furthermore, the substitution of a pyrophosphate bond for a scissile phosphodiester bond in one strand completely inhibited digestion in this strand without any decrease of the rate of hydrolysis of the unmodified strand. In contrast to EcoRII endonuclease, which recognizes the same DNA sequence, in the case of MvaI endonuclease substrate recognition is possible in a wide range of conformational, electronic and hydrophobic alterations within the recognition site.
Purification and characterization of a β‐glucosidase from Evernia prunastri
Authors: YAGÜE Ernesto , ESTÉVEZ M. Pilar
Published: March 2005

Intracellular β‐glucosidase from Evernia prunastri has been purified to homogeneity using anion exchange on DEAE‐Seaphadex A‐50, and gel filtration chromatography on Sephadex G‐100 and Sepharose 6B. The purified β‐glucosidase showed a single protein band on native electrophoresis and its isoelectric point was at pH 3.12. The molecular mass, calculated from its partition coefficient on the Sepharose 6B column, was 311 kDa, being composed of several subunits of 60 and 70 kDa. The highest activity of this enzyme was attained at pH 4.0 and 60°C. The enzyme showed strong resistance to thermal inactivation. Its activation energy was about 15 kJ/mol. Cellobiose, salicin, and p‐nitrophenyl β‐D‐glucoside, but not carboxymethylcellulose, were hydrolyzed by the enzyme, following substrate inhibition kinetics. The purified β‐glucosidase was considered a true cellobiase because of its great affinity towards cellobiose. Cellobiose inhibition does not seem to be a physiological phenomenon. Glucose inhibited enzyme activity in a competitive way (Ki= 1.26 mM). Fe3+ and Co2+ inhibited activity notably. Hg2+, Cu2+ and EDTA were practically ineffective. Even 200 mM gluconolactone did not affect enzyme activity.
Purification and characterization of rat‐liver cytosolic epoxide hydrolase
Authors: SCHLADT Ludwig , HARTMANN Renate , WÖRNER Walter , THOMAS Helmut , OESCH Franz
Published: March 2005

Rat liver cytosolic epoxide hydrolase has been purified and characterized.

The enzyme was purified from tiadenol‐induced rat liver 540‐fold with respect to trans‐stilbene oxide as a substrate. Similar purification was obtained with the substrates trans‐β‐ethyl styrene oxide and styrene 7,8‐oxide, the specific activities decreasing in the order trans‐β‐ethyl styrene oxide > styrene 7,8‐oxide > trans‐stilbene oxide. The enzyme exerts highest activity at pH 7.4. Km and Vmax of the pure enzyme for trans‐stilbene oxide were 1.7 μ and 205 nmol × min−1× mg protein−1 respectively. With trans‐stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol < methanol < acetone < isopropanol = N,N‐dimethyl formamide < acetonitrile < tetrahydrofuran. The native enzyme, with a molecutar mass of 120 kDa, consists of two 61‐kDa subunits.

Digestion of rat liver cytosolic and microsonal epoxide hydrolase by. three proteases resulted in markedly different peptide maps.

Western‐blot analysis with antiserum agnnst rat liver cystosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofiberate‐iduced rats. No cross‐reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes.

A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig. Syrian hamster and New Zealand white rabbit but not with that of green monkey.
A rapid method of epitope mapping
Authors: LORENZO Fredéric , JOLIVET André , LOOSFELT Hugues , THU VU HAI Mai , BRAILLY Sylvie , PERROT‐APPLANAT Marline , MILGROM Edwin
Published: March 2005

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C‐terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C‐terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action.

This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1‐60, 101‐110, 295‐325 and 370‐396. There were no antibodies directed against the DNA‐binding or the steroid‐binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross‐reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1‐60).

The 79‐kDa form (‘subunit A’) of the receptor was shown to lack the two more N‐terminally localized immunogenic domains (amino acids 1‐60 and 101‐110). The 65‐kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N‐terminal part of the protein.
Protease‐nicked O‐toxin of Clostridium perfringens, a new memnrane probe with no cytoltic effect, revcals two classes of cholesterol as toxin‐binding sites on sheep erythrocytes
Authors: OHNO‐IWASHITA Yoshiko , IWAMOTO Machiko , MITSUI Ken‐ichiro , ANDO Susumu , NAGAI Yoshitaka
Published: March 2005

A nicked O‐toxin (Co), Obtaned by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20°C. Neither electron micreoscopic evidence for the formation of arc‐ and ring‐shaped structures on the membrane non toxin‐stimulated influx of extracellular Ca2+ are detected in Co‐treated cells below 20°. Thus, event(s) in the lytic process are responsible for the temperature deoebdency of hemolysis, which is also supported by the observation that Co requires higher Arrhenius activation energy for hemolysis than the native toxin. Using Co as a probe due to its high affinity ofr membrane cholesterol without causing any obvious membrane change, we demonstrated the possible existence of high‐and low‐affinity sites for o‐toxin on sheep erythrocytes. both binding sites desappear by simulataneous treatment of the cell with sublytic doses of digitonin. Furthermore, Co Binds only to cholesterol among the chloroform/methanol‐ extractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them. these results strongly suggest that cholesterol is and essential component of the both high‐ and low‐affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.
Enzymes as biosensors
Authors: RICARD Jacques , BUC Jean
Published: March 2005

When a free enzyme exists under different conformations that ‘slowly’ isomerize during the conversion of a substrate into a product, the corresponding ‘slow’ relaxation component may interfere with the steady‐state component. The apparent steady‐state rate that may be measured under these conditions is called the meta‐steadystate rate for it refers to the existence of meastable states of the enzyme during the reaction. By contrast to the real steady‐state rate, the meta‐steady‐state rate is dependent upon the initial state of the enzyme, that isonthe respective concentrations of the free enzyme forms.

The simplest model that may display this type of behaviour is the mnemonical model. For a fixed concentrationof the last product of the reaction sequence the meta‐steady state is different depending on that concentration being reached by an increase or a decrease of a previous concentration. This means that the meta‐steady‐state rate describes a hysteresis loop as the product concentrations is increased and decreased. Owing to the existence ofmetastable states, the enzyme system behaves as a biosensor that is able to detect both a concentration andthe direction of a concentration change.

The existence of the hysteresis loop of the meta‐steady‐state rate implies that the two free enzyme forms display hysteresis as well. A chemical potential, called the sensing potential, is specifically associated with the ‘perception’of the direction of the thermodynamic force generated by the decrease or the increase of the concentration of the ligand that binds to one of the enzyme conformations. The sensing potential of the enzyme conformer that does not bind the product increase and reaches a plateau as the chemical potential of that product is raised. Alternatively the sensing potential of the other conformer vanishes at low and high chemical potentials of the product and is significant for intermediate chemical potentials.

Enzymes that display very slow conformation changesmay thus be viewed as elementary sensor devices.
Histone H1 heterogeneity in the midge, Chiromomus thummi
Authors: HOYER‐FENDER Sigrid , GROSSBACH Ulrich
Published: March 2005

Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I‐1, II‐1, II‐2, 11‐3, III‐1, III‐2, and III‐3, according to the order of migration in two steps of preparative electrophoresis.

The amino acid compositions are similar to those of other H1 histones. Subtractions I‐1 and II‐1 were found to contain one methionine and two tyrosine residues, II‐2 contained two methionine and three tyrosine residues, and III‐1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I‐1 and II‐1 a chain length of about 252 amino acids was estimated.

Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subtraction of histone H2A disappeared after this treatment Most and very probably all subfractions are thus H1 sequence variants.

Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I‐1 and at least some of the other variants.

H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison discriminates the group of C. thummi H1 histones clearly from Drosophila and calf thymus H1. Limited trypsin digestion yielded a protected peptide of uniform size in six of the seven variants which was considerably smaller than the protected central domain of calf thymus H1.

Two other species of Chironomidae, C. pallidivittatus and Glyptotendipes barbipes were found to contain five and three H1 subfractions, respectively.

The results provide a basis for the localization of H1 variants in specific chromatin regions within the giant chromosomes by means of monoclonal antibodies and may lead to an understanding of the functional significance of H1 heterogeneity.
Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin β subunit tetramers
Authors: CRAESCU Constantin T. , MISPELTER Joël
Published: March 2005

Isolated β chains from human adult hemoglobin at millimolar concentration are mainly associated to form β4 tetamers. We were able to obtain relevant two‐dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr∼ 66000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated β(CO) chains in solution and in β(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu‐141 (H19) in isolated β chains. Comparison of the assigned resonances of conserved amino acids in α chains, β chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val‐Ell, and on Leu‐H19 and Phe‐G5 position on the proximal side.
Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine‐plus‐cytosine contents
Authors: HORINOUCHI Sueharu , FUKUSUMI Shoji , OHSHIMA Toyoji , BEPPU Teruhiko
Published: March 2005

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and chgaracterized, weh have cloned two additional genes, amyB, and amyC, coding for amylases of this thermophile, into Escherichia coil and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coil promoters. Almost all activity was detected in the intracellular fraction in the E. coil cells.The molecular mass and NH2‐terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coil transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59000). The optimal temperatures for AmyB and AmyC activities on soluble strach were 80oC and 70oC, respectively. Both AmyB and AmyC showed a pH optimum of 5.5 AmyB and AmyC showed a different pattern of strach hydrolysis when examined by thinlayer chromatography. Some homology in the amino sequences with the functional regions of Taka‐amylase A was found in both AmyB and AmyC.

The condon usage in the amyA., amyB and amyC genes was highly biased, which reflects the fact that the guanine‐plus‐cytosine (G+C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the condons was non‐random; the G+C content of the first position of condons is significantly high, whereas that of the third position is somewhat low. In addition, condons consisiting only of A and T were preferntially used in this thermophile.
Complete nucleotide sequence of the ribosomal ‘A’ protein operon from the archaebacterium, Halobacterium halobium
Authors: ITOH Takuzi
Published: March 2005

DNA fragments cloned from the halophilic archaebacterium Halobacterium halobium including the gene for ribosomal ‘A’ protein (HhaL20) were sequenced. Sequence and transcript analyzes indicated that the ‘A’ protein message (ORFC) was co‐transcribed with two neighbouring open reading frames (ORFA and B) as a single unit. Comparison of the amino acid seuqences deduced from ORFA and B with those from other organisms demonstrated that ORFA encoded the protein homologous to the ribosomal protein L8 of Halobacterium cutirubrum (HcuL8) and LI of Escherichia coli (ELI), while ORFB encoded the protein homologous with the human ribosomal protein PO. The sequences which were able to facilitate mRNA · 16S‐rRNA hybridization were found about 10 ‐ 20 nucleotides upstream of the initiation codon ATG of each of the three ORFs. The 5′‐ and 3′‐ends of the operon were mapped with nuclease SI. A putative promoter (A+T‐rich) and termination signal (T‐rich) were found to be present within the 5′‐ and 3′‐flanking sequence.
Cardiac troponin I, isolated from‐bovine heart, contains two adjacent phosphoserines
Authors: SWIDEREK Kristine , JAQUET Kornelia , MEYER Helmut E. , HEILMEYER Ludwig M. G.
Published: March 2005

Bovine cardiac troponin containing approximately 3 mol P/mol protein could be separated into its subunits without loss of phosphate. Troponin I and troponin T each contain about 1.5 mol P/mol protein. In troponin I two phosphorylated serine residues could be localized in the N‐terminal region by conversion of phosphoserine to S‐ethylcysteine. They are located in adjacent positions in the following sequence:‐Arg‐Arg‐Ser(P)‐Ser(P)‐Ala‐Asn‐Tyr‐Tyr‐Arg‐Ala‐Tyr‐Ala‐Thr‐Glu‐Pro‐His‐Ala‐Lys. This sequence shows that the first phosphoserine residue in bovine cardiac troponin I occupies a homologous position to phosphoserine‐20 of rabbit cardiac troponin I.
Studies on the effect of stigmatellin derivative on cytochrome band the Rieske iron‐sulfur cluster of cytochrome c reductase from bovine heart mitochondria.
Authors: OHNISHI Tomoko , BRANDT Ulrich , von JAGOW Gebhard
Published: March 2005

Stimatellin and its derivatives represent a third class of Q0 site inhibitor beside the hydroxyquinone derivatives and the E‐β‐methoxyacrylate (MOA) inhibitor [von Jagow and Link (1986) Method Enzymol 126., 253‐271]. The stigmatellins consist of a chromone ring system connected to an substituted alkenyl side chain. Alteration in the side chain, i.e. saturation of the C=C double bonds, shift of methoxy group or loss of the methyl group, specifically affect the binding characterstics. Besides changing the red shift spectrum of reduced cytochrome b566 and the ERR spectrum of the Rieske iron‐ sulfur clusterm, the side chain alteration diminish the binding affnity and the extent of the midpoint potential shift of the iron‐sulfur protein. Thus, the side chin of the molecule makes an essentils contribution to the binding energy and is hot necessary solely for partitioning the molecule into the hydrophobic phase, as assumed so far.
Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle
Authors: PITCHER Julie , SMYTHE Carl , COHEN Philip
Published: March 2005

Purified preparations of‐glycogen synthase are a complex of two proteins, the catalytic subunit of glycogen synthase and glycogenin, present in a 1:1 molar ratio [J. Pitcher, C. Smythe, D. G. Campbell & P. Cohen (1987) Eur. J. Biochem. 169, 497‐502]. This complex has now been found to contain a further glucosyltransferase activity that catalyses the transfer of glucose residues from UDP‐Glc to glucosylated‐glycogenin. The glucosyltransferase, which is of critical importance in forming the primer required for de novo glycogen biosynthesis, is distinct from glycogen synthase in several ways. It has an absolute requirement for divalent cations, a 1000‐fold lower Km for. UDP‐Glc and its activity is unaffected by incubation with UDP‐pyridoxal or exposure to 2 M LiBr, which inactivate glycogen synthase by 95% and 100%, respectively.

The priming glucosyltransferase and glycogen synthase activities coelute on Superose 6, and the rate of glycosylation of glycogenin is independent of enzyme concentration, suggesting that the reaction is catalysed intramolecularly by a subunit of the glycogen synthase complex. This component has been identified as glycogenin, following dissociation of the subunits in 2 M LiBr and their separation on Superose 12. The glycosylation of isolated glycogenin reaches a plateau when five additional glucose residues have been added to the protein, and digestion with a‐amylase indicates that all the glycogenin molecules contain at least one glucosyl residue prior to autoglucosylation. The priming glucosyltransferase activity of glycogenin is unaffected by either glucose 6‐phos‐phate or by phosphorylation of the catalytic subunit of glycogen synthase. The mechanism of primer formation is discussed in the light of the finding that glycogenin is an enzyme that catalyses its own autoglucosylation.
Assambley of amber fragment of the β subunit of Escherichia coli RNA polymerase
Authors: GLASS Robert E. , RALPHS Nicola T. , FUJITA Nobuyuki , ISHIHAMA Akira
Published: March 2005

Immunoblotiing of size‐ seperated whole cell protein permitted the study of protein‐protein interaction. Briefly, protein obtained from cleared cell lysates of Escherichia coli were seperated by glycerol gradient centrifugation and analysed by blooting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N‐terminla amber fragment of the β subunit of E. coli RNA polymerase ranging in size between 97% and 23% the lenght of the intact β polypeptide (1342 amino acids). In this way, we have been able to define regions on the β polypeptide involved in the assembly of RNA polymerase.
Characterization of the inducible polyamine transporter in bovine lymphocytes
Authors: KAKINUMA Yoshimi , HOSHINO Kenji , IGARASHI Kazuei
Published: March 2005

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 uM, 0.38 uM and 0.23 uM in that order. The uptake activity was inhibited by carbonyl cyanide m‐chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as α‐difluoromethyl‐ornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 uM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane‐potential‐dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.
Purification of RNA 3′‐terminal‐phosphate cyclase from HeLa cells
Authors: VICENTE Oscar , FILIPOWICZ Witold
Published: March 2005

RNA S′‐terminal phosphate cyclase has been purified about 6000‐fold to near homogeneity from HeLa cells. The purified protein is a single polypeptide with an Mr of 38000‐40000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0‐9.0. In the presence of Mg 2+ and ATP this enzyme catalyzes the conversion of a 3′‐phosphate group into the cyclic 2′,3′‐phosphodiester at the 3′ end of RNA, through formation of a covalent cyclase‐AMP intermediate. GTP, CTP and UTP (but not dATP or ADP) can also function as cofactors in the cyclization reaction, although less efficiently (apparent Km values for ATP and GTP are 6 μM and 200 μM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [α‐32P]NTPs.
Citric‐acid cycle, 50 years on
Authors: THAUER Rudolf K.
Published: March 2005

Many anaerobic bacteria can completely oxidize organic matter to CO2 with either sulfur, sulfate, or protons as electron acceptor. The sulfur‐reducing bacteria and one genus of sulfate reducers use a modified citric‐acid cycle with a novel anaplerotic sequence as pathway of terminal respiration. All other anaerobes use an alternative pathway, in which carbon monoxide dehydrogenase is a key enzyme and in which acetyl‐CoA is cleaved into two C1 units at the oxidation level of CH3 OH and CO. Thus almost 50 years after the discovery of the citric acid cycle by Hans Krebs in 1937, a second pathway for acetyl‐CoA oxidation was found.
Storage of sialic acid‐containing carbohydrates in the placenta of a human galactosialidosis fetus
Authors: van PELT Johannes , VAN KUIK J. Albert , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , VAN DIGGELEN Otto P. , GALJAARD Hans
Published: March 2005

From the placenta of a human fetus with galactosialidosis, detected by prenatal diagnosis, sialyloligosaccharides were isolated by successively gel‐permeation chromatography on Bio‐Gel P‐6, anion‐exchange chromatography on Mono Q and high‐performance liquid chromatography on Lichrosorb‐NH2. 16 sialic acid‐containing N‐glycosidic N‐acetyllactosamine type of structures were identified by sugar analysis and 500‐MHz 1H‐NMR spectroscopy. The fully sialylated oligosaccharides differ from each other in the type of branching (mono‐, di‐, tri‐, tri'‐, and tetra‐antennary) or sialic acid linkage types (α2–3/α2–6). The isolated carbohydrates, including six novel structures, can be presented as follows (the numerals in a column represent one compound).
Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii
Authors: SCHUSTER Gadi , TIMBERG Rina , OHAD Itzhak
Published: March 2005

The turnover of photosystem‐II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae‐CP‐29 and light‐harvesting‐complex‐II polypeptides and the water‐oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis‐saturating light (2500 W m2 for 90 min) at 25°C. While 80% of photosystem‐II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold‐labeled whole‐cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition.

We propose that the light‐dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem‐II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.
Isolation and structural characterization of low‐molecular‐mass monosialyl oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: VAN HALBEEK Herman , BREG Jan , VLIEGENTHART Johannes F. G. , KLEIN André , LAMBLIN Geneviéve , ROUSSEL Philippe
Published: March 2005

The carbohydrate chains of the respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low‐molecular‐mass, monosialyl oligosaccharide‐alditols were isolated by anion‐exchange chromatography and fractionated by consecutive straight‐phase high‐performance liquid chromatography (HPLC) on a silica‐based alkylamine column, and reverse‐phase HPLC on a silica‐based octadecyl column, respectively. The structures of the oligosaccharide‐alditols were determined by 500‐MHz 1H‐NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are:

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23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631‐642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643‐654]. The sialic acid residue was found to occur either in α(2→3)‐ or α(2→6)‐linkage to a galactosyl residue or in a(2→6)‐linkage to GalNAc‐0l.
Induction of S100 protein in 3T3‐L1 cells during differentiation to adipocytes and its liberating by lipolytic hormones
Authors: KATO Kanefusa , SUZUKI Fujiko , OGASAWARA Nobuaki
Published: March 2005

When confluent cultures of cloned mouse 3T3‐L1 cells were differentiated to adipocytes by three days of treatment with a combination of 0.5 μM dexamethasone and 0.5 mM 1‐methyl‐3‐isobutylxanthine, the S100 protein content in the cells increased markedly, as determined by a sensitive immunoassay system. The S100 protein induced in the cell was the αα form (S100a0), which is the predominant form of S100 protein in mouse adipose tissue. The S100a0 concentration in preadipocytes was about 1—3 ng/mg protein, while the concentration in differentiated adipocytes was 60—200 ng/mg protein. The immunoblotting test of the crude extract of adipocytes confirmed that the immunoreactive substance in the cells was the α subunit of S100 protein. The treatment with 1‐methyl‐3‐isobutylxanthine or dexamethasone alone neither elicited the S100 protein induction nor triacylglycerols accumulation in the cells. The accumulation of triacylglycerols in the cells was always preceded by the induction of S100a0 protein under conditions where the differentiation to adipocytes was elicited. The induction of S100a0 protein and accumulation of triacylglycerols in the cells treated with dexamethasone and 1‐methyl‐3‐isobutylxanthine were inhibited by the addition of antimicrotubular drugs, colchicine and vinblastine, but not by cytochalasin B, an antimicrofilament drug. S100a0 protein in 3T3‐L1 adipocytes was released by incubation with a lipolytic hormone, adrenocorticotropic hormone or catecholamines, in a cyclic‐AMP‐dependent manner as observed with rat epididymal fat pads [Biochim. Biophys. Acta (1986) 889, 84–90]. These results also suggest that S100 protein may participate in the function of adipocytes.
Purification of transfer RNA (m5U54)‐methyltransferase from Escherichia coli
Authors: NY Tor , LINDSTRÖM Pär H. R. , HAGERVALL Tord G. , BJÖRK Glenn R.
Published: March 2005

tRNA (m5U54)‐methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S‐adenosyl‐L‐methionine to transfer ribonucleic acid (tRNA) and thereby forming 5‐methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800‐fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)‐methyltransferase was used to amplify genetically the production of this enzyme 40‐fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48–57‐kDa RNA‐protein complex. All the polypeptides seem to be related to the 42/41‐kDa polypeptides previously identified as the tRNA (m5U54)‐methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N‐terminal sequence of the 42‐kDa polypeptide was found to be: Met‐Thr‐Pro‐Glu‐His‐Leu‐Pro‐Thr‐Glu‐Gln‐Tyr‐Glu‐Ala‐Gln‐Leu‐Ala‐Glu‐Lys‐. The tRNA (m5U54)‐methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S‐adenosyl‐L‐methionine are 80 nM and 17 μM, respectively.
The binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase
Authors: SARTORIUS Christian , DUNN Michael F. , ZEPPEZAUER Michael
Published: March 2005

We have studied the binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase [Co(II)‐LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10‐phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10‐phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10‐phenanthroline to Co(II)‐LADH is about 60 s−1. This is about a third of the value (169 s−1) determined for native horse‐liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471–480]. For cadmium(II)‐substituted horse‐liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10‐phenanthroline increased to 590 s−1. These findings demonstrate that the rate‐limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10‐Phenanthroline is shown to bind to the Co(II)‐LADH · NAD+ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD+. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD+ that involves a slow intermediary step.
Ribosomal protein interactions in yeast
Authors: SÁENZ‐ROBLES M. Teresa , VILELLA M. Dolores , PUCCIARELLI Graciela , POLO Florentino , REMACHA Miguel , ORTÍZ Blanca L. , VIDALES Fernando J. , BALLESTA Juan P. G.
Published: March 2005

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44′ and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM‐cellulose and DEAE‐cellulose columns and from reverse‐phase HPLC columns. Moreover, immunoprecipitation using anti‐L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic‐adsorbed antibodies specific for one of the components in the L15–acidic‐protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross‐linked to the acidic proteins in solution.

These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276–281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El‐Baradi et al. (1987) J. Mol. Biol. 195, 909–917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.
Expression of cytochrome P‐450 isozymes in the liver of hypophysectomized rats
Authors: LEMOINE Antoinette , MARIE Sandrine , CRESTEIL Thierry
Published: March 2005

Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P‐450 species. In adult males, hypophysectomy caused a decrease of total P‐450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity.

When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti‐(phenobarbital‐induced P‐450) which was not observed with a monoclonal antibody of anti‐(phenobarbital‐induced P‐450).

These microsomal preparations also reacted with an antibody raised against a developmentally regulated P‐450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction.

Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P‐450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital‐inducible mRNA for UDP‐glucuronosyltransferase.

In contrast, P‐450 mRNA induced by pregnenolone 16α‐carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone‐16δ‐carbonitrile‐induced P‐450 mRNA accumulation.

Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P‐450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P‐450s.
Alteration of the genes for glycophorin A and B in glycophorin‐A‐deficient individuals
Authors: RAHUEL Cécile , LONDON Jacqueline , VIGNAL Alain , CHERIF‐ZAHAR Baya , COLIN Yves , SIEBERT Paul , FUKUDA Minoru , CARTRON Jean‐Pierre
Published: March 2005

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood‐group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution.

The lack of glycophorin A is associated with the rare phenotype En(a–), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a–) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a –) variants and in the En(a +) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM‐B, composed of the N‐terminal portion of a blood group M‐type glycophorin A and of the C‐terminal portion of glycophorin B. The determination of the 5′ and 3′ limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.
Mycolic acids of Mycobacterium aurum
Authors: LANÉELLE Marie‐Antoinette , LACAVE Charlotte , DAFFÉ Mamadou , LANÉELLE Gilbert
Published: March 2005

Mycobacterium aurum (type strain) was analyzed for its mycolate content. Three types of mycolates were identified: di‐unsaturated, oxo and dicarboxy mycolates, each type being constituted by two subtypes. The acid released by pyrolysis was identified as docosanoic acid. By use of mass spectrometry and oxidation techniques, the structures of these six subtypes of mycolates were elucidated. They contain Z and E double bonds, the latter having an adjacent methyl branch. No cyclopropane ring was observed. All the methyl branches occurring in these mycolates derived from methionine, the methyl‐branched chiral center adjacent to the double bond having an R configuration.

The structures of the major di‐unsaturated mycolates suggest that they cannot be the precursors of oxo mycolates.

The amount of long‐chain secondary alcohol (2‐octadecanol) obtained from the whole cells was found to be much greater than that expected from hydrolysis of wax ester mycolate (ester of 2‐octadecanol and dicarboxymycolic acid). Further investigations showed that 2‐octadecanol was also present in triacylglycerols, esterifying the ω‐carboxyl group of long‐chain fatty acids structurally related to dicarboxy‐mycolates.
Step‐wise progression of mammalian 10‐kb DNA replication intermediates to mature chromatin
Authors: LÖNN Ulf , LÖNN Sigrid
Published: March 2005

In mammalian DNA synthesis the primary replication intermediates are joined to larger intermediates. After the joining process is complete one can detect a distinct stage called the post‐elongation stage. Furthermore a 10‐kb DNA1 population is detected before the post‐elongation stage whereas a 10‐kb DNA2 population is part of this stage DNA. When cells are treated with 3‐aminobenzamide, an inhibitor of poly(ADP‐ribose) synthetase, an altered post‐elongation‐stage DNA was detected, which does not contain 10‐kb fragments. The step(s) affected by 3‐aminobenzamide prevents the appearance of 10‐kb DNA in the post‐elongation stage. The drug effect is reversible with the appearance of 10‐kb DNA in the post‐elongation stage when the cells are washed free of drug. Hence there is a step‐wise progression from 10‐kb DNA, via the post‐elongation stage, to mature chromatin.
Tubulin oligomers and microtubule oscillations
Authors: LANGE Gudrun , MANDELKOW Eva‐Maria , JAGLA Annette , MANDELKOW Eckhard
Published: March 2005

Several types of non‐equilibrium phenomena have been observed in microtubule polymerization, including dynamic instability, assembly overshoot and oscillations. They can be interpreted in terms of interactions between tubulin subunits (=α, β heterodimers), microtubules, and a third state, oligomers, which represent intermediates between microtubule disassembly and the regeneration of assembly‐competent subunits by GTP. Here we examine the role of oligomers by varying conditions that stabilize or destabilize microtubules and/or oligomers. By varying their ratio one can drive tubulin assembly either into steady‐state microtubules or oligomers. These regimes of assembly conditions are separated by a region where microtubules oscillate. The oscillations can be simulated by computer modelling, based on a reaction scheme involving the three states of tubulin and nucleotide exchange on tubulin subunits, but not on microtubules or oligomers.