bluuebunny/crossref_metadata_2025_split huggingface dataset:
Demonstration of T‐Transferase Deficiency in Tn‐Polyagglutinable Blood Samples
Authors: CARTRON Jean‐Pierre , ANDREU Georges , CARTRON Jeannine , BIRD George W. G. , SALMON Charles , GERBAL Alain 
Published: March 2005

The serum and red cell membranes from seven Tn individuals have been tentatively characterized for the UDPgalactose: N‐acetyl‐d‐galactosamine‐β‐d‐galactosyltransferase and UDPgalactose: N‐acetyl‐d‐glucosamine‐β‐4‐d‐galactosyltransferase activities using p‐Nitrophenyl‐2‐acetamido‐2‐deoxy‐α‐d‐galactopyranoside and p‐Nitrophenyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside respectively as low molecular weight acceptors. In five cases, Tn‐positive and Tn‐negative red cells were at first separated by Polybrene differential aggregation. The following conclusions have been drawn.

The β‐3‐d and β‐4‐d‐galactosyltransferases activities are found in serum and red cell membranes from all normal individuals.
Polybrene‐positive cells (normal sialic acid content) from Tn bloods have either normal or higher β‐3‐d and β‐4‐d‐galactosyltransferases activities.
Polybrene‐negative cells (low sialic acid content) from Tn bloods have a selective deficiency in β‐3‐d‐galactosyltransferase (T‐transferase) activity, but normal or even increased β‐4‐d‐galactosyl‐transferase activity.
The serum from all Tn individuals behaves like normal sera in respect of the two galactosyl‐transferase activities. The serum may also be used as source of enzyme for conversion in vitro of Tn to T‐reactive erythrocytes.

The above data strongly support the view that Tn‐polyagglutination results from a somatic mutation in stem cells of haematopoietic tissue which involves a single genetic step in red cell glycoprotein synthesis. It also provides further evidence of a dual population of erythrocytes in each Tn blood sample and suggests that T‐transferase found in serum is produced in unidentified cells of the organism. No difference has been noticed between apparently healthy Tn donors and Tn patients.
Kinetic Evidence for Two Sites in the Inhibition by Diuron of the Electron Transport in the bc1 Segment of the Respiratory Chain in Saccharomyces cerevisiae
Authors: CONVENT Bernard , BRIQUET Michel , GOFFEAU André 
Published: March 2005

A kinetic method based on the measurement of NADH oxidase activity in yeast submitochondrial particles incubated in the simultaneous presence of an inhibitor of high affinity such as antimycin and an inhibitor of low affinity such as 3(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron) permits estimation of two distinct dissociation constants for diuron, Kd.1= 3.2 μM and Kd,2= 21.5 μM.
To take into account the fact that these inhibitors do not act on the rate‐limiting step of the NADH oxidase activity, the Kröger and Klingenberg kinetic model [Eur. J. Biochem. (1973) 39, 313–323] has been applied using a new method for estimating the maximal activities of the NADH: Q reductase activity (Vred= 280 nmol O × min−1× mg −1) and of the QH2: O2 oxidase activity (Vox= 850 nmol O × min−1× mg−1) (Q = ubiquinone). These latter values permit the estimation of two distinct dissociation constants for another weakly bound inhibitor, n‐heptyl‐hydroxyquinoline‐N‐oxide: Kh,1= 0.23 μM and Kh,2= 5 μM.
Only one dissociation constant, Kd= 23 μM for diuron is sufficient to characterize the ‘extra‐reduction’ of cytochrome b566.
Even though diuron does not induce the ‘red‐shift’ of cytochrome b562, the combined addition of suboptimal concentrations of n‐heptyl‐hydroxyquinoline‐N‐oxide and diuron, permits estimation of a single dissociation constant which is lower than 6.6 μM diuron for a site which can bind either diuron or n‐heptyl‐hydroxyquinoline oxide but which will yield a ‘red‐shift’ of cytochrome b562 when occupied by n‐heptyl‐hydroxyquinoline‐N‐oxide only.
The Effect of Salt Concentration on the Inhibition of Protein Synthesis by Double‐Stranded RNA
Authors: BAGLIONI Corrado , LENZ Jack R. , MARONEY Patricia A. 
Published: March 2005

Double‐stranded RNAs (dsRNA) such as poly(I) · poly(C) inhibit protein synthesis in reticulocyte lysate incubated with low concentrations of potassium salts more than in lysate incubated with higher concentration of salt. The inhibition of protein synthesis by dsRNA takes place after an initial lag. In the presence of high salt concentrations the lag period before the inhibition occurs is considerably increased. The maximum inhibitory concentration of poly(I) · poly(C) remains the same at all salt concentrations tested; high concentrations of dsRNA are not inhibitory under different ionic conditions.The inhibition of protein synthesis by dsRNA is due to a loss of the Met‐tRNAf binding activity of native 40 S ribosomal subunits. However, lysates incubated with dsRNA at high salt concentrations retain Met‐tRNAf binding activity. Initiation factors(elF) isolated from ribosomes of lysates incubated under different ionic conditions have been used to determine the activity of the initiation factor, elF‐2, which catalyzes the binding of Met‐tRNAf to 40‐S subunits. The initiation factors from lysates incubated with poly(I) · poly(C) at low salt concentrations are less active in binding Met‐tRNAf and GTP than those from lysates incubated at high salt concentrations. Ribosomes washed with 0.5 M KCl obtained from these lysates are equally active in binding Met‐tRNAf to 40‐S subunits when supplemented with fresh initiation factors, indicating that the loss in Met‐tRNAf binding activity is due to a loss in initiation factor activity and not to a loss of any 40‐S subunit activity.The decreased inhibitory activity of dsRNA at high salt concentrations is due both to a delay in the formation of a dsRNA‐activated inhibitor, and to a reduced effect on protein synthesis of the inhibitor.
A New Method of Chemical Modification of N6‐Amino Group in Adenine Nucleotides with Formaldehyde and a Thiol and Its Application to Preparing Immobilized ADP and ATP
Authors: YAMAZAKI Yoshimitsu , SUZUKI Hideo 
Published: March 2005

Reaction of AMP with formaldehyde and 3‐mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6‐[(2‐carboxyethyl)thiomethyl]‐AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6‐[(2‐carboxyethyl)thiomethyl]‐9‐methyladenine (III) and N6, N6‐bis[(2‐carboxyethyl)thiomethyl]‐9‐methyladenine (IV) which were synthesized from 9‐methyladenine in the same way as for derivatives I and II.ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6‐[(2‐carboxyethyl)thiomethyl]‐ADP (V), N6‐[(2‐carboxyethyl)thiomethyl]‐ATP (VI), N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐ADP (X) and N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3‐diaminopropane with a water‐soluble carbodiimide to give the corresponding amino derivatives, N6‐{[N‐(3‐aminopropyl)carbamoylethyl]thiomethyl}‐ADP (VIII), N6‐{[N‐(3‐aminopropyl)carbamoylethyl]thiomethyl}‐ATP (IX), N6,N6‐bis{[N‐(3‐aminopropyl)carbamoyleythyl]thiomethyl}‐ADP (XIII), and N6,N6‐bis{[N‐(3‐aminopropyl)carbamoyletethyl]thiomethyl}‐ATP (XIV), which were further bound to CNBr‐activated dextran to give new polymer‐bound derivatives of ADP and ATP.These free and bound derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran‐bound VIII, and dextran‐bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran‐bound IX, and dextran‐bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.
Translation of MS2 RNA in vitro in the Absence of Initiation Factor IF‐3
Authors: ZIPORI Pnina , BOSCH Leendert , VAN DUIN Jan 
Published: March 2005

An Escherichia coli cell‐free translational system, deprived of initiation factor IF‐3, has been used to study the role of the factor in protein synthesis. In this system, 30‐S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process prermits formation of functional complexes which can carry out protein synthesis in the complete absence of IF‐3.The translational products, obtained in the absence of IF‐3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.
Effect of Poly(ADP‐ribose) Formation on DNA Synthesis in Chick‐Embryo‐Liver Nuclei
Authors: TANIGAWA Yoshinori , KITAMURA Akihide , KAWAMURA Masao , SHIMOYAMA Makoto 
Published: March 2005

When chick‐embryo‐liver nuclei were incubated with NAD, incorporation of [3H]dTMP into the acid‐insoluble fraction by either endogenous or exogenous DNA polymerase was enhanced. An NAD concentration of 1 mM was sufficient for maximal stimulation of DNA synthesis. Furthermore, the stimulation of DNA synthesis was proportional to the amounts of poly(ADP‐ribose) formed in the nuclei.With a constituted system for DNA synthesis, it was observed that when small amounts of DNA were employed as template, the DNA synthesis increased in the presence of NAD‐treated extract of the nuclei compared to that in the presence of the untreated extract. The NAD‐treated extract is heat‐stable for enhancement of DNA synthesis. The nuclease activity as catalyzed by exogenous pancreatic DNase I in the presence of NAD/heat‐treated extract was higher than that in the presence of heat‐treated extract, when small amounts of [3H]DNA were employed as substrate.The DNAs isolated from the [3H]thymidine‐labelled nuclei were prepared from chick embryo liver, incubated with NAD, and then analyzed by ultracentrifugation in an alkaline sucrose gradient. We found the formation of smaller fragments by endonucleolytic cleavage to be enhanced.These results strongly suggest that the stimulation of DNA synthesis by poly(ADP‐ribose) formation is due to an increase in accessibility of nuclease to DNA as a result of the template activation.
The Poly(glycosyl) Chains of Glycoproteins.
Authors: KRUSIUS Tom , FINNE Jukka , RAUVALA Heikki 
Published: March 2005

Glycopeptides with complex carbohydrate structure were isolated from delipidated human erythrocyte membranes after digestion with pronase. The poly(glycosyl)peptides isolated (apparent molecular weight 4000–13 000) are suggested to contain 20–70 sugar residues in an alkali‐stable saccharide chain linked through N‐acetylglucosamine to asparagine. The main sugar components are galactose and N‐acetylglucosamine, which together account for 80% of total sugars. That the compounds isolated are glycopeptides and not glycolipids is concluded from the following findings: only trace amounts of glucose and fatty acids were present, and no long‐chain (sphingosine) bases could be detected; on the other hand, the amounts of mannose and amino acids found are compatible with an N‐glycosidic poly(glycosyl)peptide structure.The structure of the poly(glycosyl)peptides was studied using methylation analysis, exoglycosidase treatments, acid hydrolysis of the native as well as the N‐deacetylated glycopeptides, and chromium trioxide oxidation. The studies indicate that the poly(glycosyl)peptides contain a repeating ‐3)galactosyl(β1–4)N‐acetylglucosaminyl(β1‐structure with branch points at the C‐6 of the galactose residues. The saccharide chains are terminated in N‐acetylglucosaminyl, galactosyl, N‐acetylneuraminyl(α2–3 and 6)galactosyl and fucosyl(α1–2)galactosyl residues, and they also contain blood group A and B determinants.
A New Biochemical Subtype of the Sanfilippo Syndrome: Characterization of the Storage Material in Cultured Fibroblasts of Sanfilippo C Patients
Authors: KRESSE Hans , VON FIGURA Kurt , KLEIN Udo 
Published: March 2005

Fibroblasts cultured from the skin of three unrelated patients with the clinical symptoms of the Sanfilippo syndrome (mucopolysaccharidosis III) accumulated intracellularly excessive amounts of heparan sulfate and showed a lengthened turnover time for this mucopolysaccharide. They exhibited, however, neither a deficiency of heparan sulfate sulfamidase or α‐N‐acetylglucosaminidase nor of any other known glycosaminoglycan‐degrading hydrolase. This new mucopolysaccharidosis was therefore designated as type C of the Sanfilippo syndrome.The abnormal heparan sulfate metabolism of Sanfilippo C fibroblasts could not be normalized by addition of crude urinary proteins or concentrated secretions from normal fibroblasts to the culture medium or by cocultivation with normal fibroblasts.The accumulated heparan sulfate was characterized by a reduced negative net charge. A small proportion of it could be adsorbed onto a cation exchange resin. It was sensitive to nitrous acid degradation under conditions where glucosamine residues with free amino groups are attacked. It is therefore suggested that the primary defect in this new mucopolysaccharidosis concerns the step which follows the hydrolysis of N‐sulfonate groups in heparan sulfate degradation.
Purification of the Elongation Factors Present in Spinach Chloroplasts
Authors: TIBONI Orsola , DI PASQUALE Giuseppe , CIFERRI Orio 
Published: March 2005

Elongation factor G (EF‐Gchl) and elongation factor Tu (EF‐Tuchl) have been purified from isolated spinach chloroplasts. On polyacrylamide gel electrophoresis the purified proteins appear to be at least 70% pure. The molecular weight has been estimated to be 77000 and 45500 for EF‐Gchl and EF‐Tuchl respectively.Chloroplast eiongation factor T (EF‐Tchl) has been only partially purified. Gel electrophoresis under non‐denturing and denaturing conditions indicate that EF‐Tchl is most probably composed of two polypeptides, one of which has an electrophoretic mobility identical to that of EF‐Tuchl.EF‐Tuchl appears to represent approximately 7% of the chloroplast soluble protein while EF‐Gchl accounts for less than 1%. Just as in the case of the bacterial factors, EF‐Tuchl appears to be in excess as compared to EF‐Gchl.Although no data were obtained on the concentration of EF‐Tchl, it may be assumed that the three elongation factors represent approximately 10% of the chloroplast soluble protein.
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: ARAI Ken‐ichi , ARAI Naoko , NAKAMURA Shun , OSHIMA Tairo , KAZIRO Yoshito 
Published: March 2005

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli.

The formation of a ternary guanine‐nucleotide · EF‐Tu · EF‐Ts complex was demonstrated by gel filtration of the T. thermophilus EF‐Tu · EF‐Ts complex on a Sephadex G‐150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli.
The dissociation constants for the complexes of T. thermophilus EF‐Tu · EF‐Ts with GDP and GTP were 6.1 × 10−7 M and 1.9 × 10−6 M respectively. On the other hand, T. thermophilus EF‐Tu interacted with GDP and GTP with dissociation constants of 1.1 × 10−9 M and 5.8 × 10−8 M respectively. This suggests that the association of EF‐Ts with EF‐Tu lowered the affinity of EF‐Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction.
Although the T. thermophilus EF‐Tu · EF‐Ts complex hardly dissociates into EF‐Tu and EF‐Ts, a rapid exchange was observed between free EF‐Ts and the EF‐Tu · EF‐Ts complex using 3H‐labelled EF‐Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides.
Based on the above findings, an improved reaction mechanism for the regeneration of EF‐Tu GTP from EF‐Tu · GDP is proposed.
Studies on the functional interchangeability of EF‐Tu and EF‐Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination.
T. thermophilus EF‐Ts could bind E. coli EF‐Tu to form an EF‐Tu (E. coli) · EF‐Ts (T. thermophilus) hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF‐Ts. On the other hand, no stable complex between E. coli EF‐Ts and T. thermophilus EF‐Tu has been isolated.
The uncoupled GTPase activity of T. thermophilus EF‐G was much lower than that of E. coli EF‐G. T. thermophilus EF‐G formed a relatively stable binary EF‐G · GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF‐G · GDP and EF‐G · GTP were 6.7 × 10−7 M and 1.2 × 10−5 M respectively. The ternary T. thermophilus EF‐G · GDP · ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF‐G.
Control of Proton Conduction by the H+‐ATPase in the Inner Mitochondrial Membrane
Authors: PANSINI Angela , GUERRIERI Ferruccio , PAPA Sergio 
Published: March 2005

Proton conduction in submitochondrial particles with various degrees of resolution of the H+‐ATPase has been studied.Proton conduction was analyzed by following the kinetics of the anaerobic decay of protons taken up by submitochondrial particles during respiratory pulses.

In EDTA submitochondrial particles proton release exhibited biphasic kinetics; both phases were depressed by collapsing aerobic Δφ.
The slow phase of proton diffusion was inhibited by oligomycin and N,N′‐dicyclohexylcarbodiimide as well as by F1 ligands like adenylyl 5′‐imidophosphate and alkyl cations. Inhibition of proton conduction by F1 ligands exhibited a sigmoidal titration curve and was synergistic with inhibition by oligomycin. Removal of the ATPase inhibitor markedly enhanced the kinetic constant of the slow proton diffusion process.
The initial rapid phase of proton diffusion was inhibited by N,N′‐dicyclohexylcarbodiimide and oligomycin but was unaffected by F1 ligands.
When the F1 moiety was removed from the particles, proton conduction became monophasic and was not any more inhibited by F1 ligands.
It is concluded that the H+‐ATPase contributes to both phases of proton conduction and that energy‐linked cooperative interaction of the F1 with the F0 moiety controls proton conductivity.

Evidence is also providëd for involvement of negatively charged groups in hydrophobic environments of the F1 moiety in proton translocation by the H+‐ATPase.
Mavicyanin, a Blue Copper Protein from Cucurbita pepo medullosa
Authors: MARCHESINI Augusto , MINELLI Martin , MERKLE Hellmut , KRONECK Peter M. H. 
Published: March 2005

The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa.
Mavicyanin contains one type‐1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (ɛ= 5000 M−1 cm−1/Cu, A280/A600= 8.0 ± 0.5, A600/A403= 7.0 ± 0.25, maximum of fluorescence emission at 335 nm.
In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz= 2.287, gy= 2.077, gx= 2.025, Az= 3.5 mT, Ay= 2.9 mT and Ax= 5.7 mT.
Like other simple type‐1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicy‐anin is readily reduced by hydroquinone or L‐ascorbic acid. Its midpoint potential E′m was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.
Methylation Sites in HeLa Cell Ribosomal Proteins
Authors: SCOLNIK Pablo A. , ELICEIRI George L. 
Published: March 2005

The methylation of HeLa cell ribosomal proteins has been studied by paper electrophoresis separation of the peptides produced after trypsin digestion of individual ribosomal proteins. Methylation in vivo was detected by 3H labeling of peptides from cells that had been incubated with a mixture of [methyl‐3H]methionine plus [35S]methionine, the incorporation of the 3H‐labeled amino acid methionine being corrected by the 35S uptake. Each methylated peptide thus represented at least one methylation site (one or more methylated amino acids and their adjacent amino acid sequence). Eleven ribosomal proteins were found to be methylated. As many as five methylation sites were present in one ribosomal protein. In several ribosomal proteins some sites were methylated when ribosome synthesis was suppressed with low levels of actinomycin D, while other sites were not. Therefore, some of the sites in a given protein were apparently methylated in mature ribosomes, while other sites were methylated during ribosome processing. The methyl‐group‐specific labeling of some sites, but not others, was suppressed by cycloheximide, thus suggesting that the methylation of some sites was dependent on protein synthesis.A cell‐free system from cultured HeLa cells is described, which reproduces the methylation pattern of ribosomal proteins in vivo in two ways: methylation was observed both in vivo and in vitro (a) in the same ribosomal proteins; and (b) in the same methylation sites of a given ribosomal protein. In addition, our data in vitro suggest that the methylation of some human ribosomal protein sites may involve a methyl donor other than S‐adenosylmethionine.
Effect of Tunicamycin on Transport of Lysosomal Enzymes in Cultured Skin Fibroblasts
Authors: VON FIGURA Kurt , REY Martin , PRINZ Roswitha , ULLRICH Kurt , VOSS Bruno 
Published: March 2005

After an initial lag phase, tunicamycin (0.12–3.6 μM) inhibits the secretion of β‐N‐acetylglucosaminidase by human skin fibroblasts. The inhibition persists for up to 30 h. Thereafter a more than fivefold‐increased rate of secretion is observed for at least 4 days. The intracellular β‐N‐acetylglucos aminidase activity decreases within 9 days in the presence of 0. 36 μM tunicamycin to 55% of that of controls. β‐N‐Acetylglucosaminidase formed in the presence of tunicamycin is only poorly recognized by fibroblasts, suggesting that tunicamycin interferes with the formation of the phosphomannosyl residues that function on β‐N‐acetylglucosaminidase as a recognition marker for the cell surface receptors present on fibroblasts. Tunicamycin (0.36–3.6 μM) inhibits endocytosis of exogenous lysosomal enzymes only after a preincubation of 24 h in the presence of the drug. Control experiments indicated that this inhibition is not due to a general inhibition of receptor‐mediated endocytosis. Incubation with tunicamycin leads furthermore to a loss of cell‐surface‐associated lysosomal enzymes.These findings indicate that tunicamycin interferes with the expression of phosphomannose receptors on the cell surface of fibroblasts and the synthesis of the phosphomannosyl residues on lysosomal enzymes after an initial lag phase. These alterations are associated with an increased accumulation of β‐N‐acetylglucosaminidase extracellularly, a loss of that enzyme from intracellular sites and from the cell surface. A similar mislocation of lysosomal enzymes in mucolipidosis II fibroblasts that is associated with a deficiency of the recognition marker on lysosomal enzymes but normal expression of the phosphomannose receptors led to the hypothesis that lysosomal enzymes undergo a secretion‐endocytosis process during their transfer into lysosomes [Hickman, S. & Neufeld, E. F. (1972) Biochem. Biophys. Res. Commun. 49, 992–999]. The present results suggest that phosphomannosyl residues within N‐glycosidically linked oligosaccharide chains on lysosomal enzymes [von Figura, K. & Klein, U. (1979) Eur. J. Biochem. 94. 347–354] function either intracellularly or on the cell surface as signal for their transfer into lysosomes.