bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of ADP‐induced platelet aggregation by reduced factor Xa
Authors: SINHA Asru K. , COLMAN Robert W. 
Published: March 2005

Treatment of blood coagulation factor Xa with insolubilized hexyl‐agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes.Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.
Protein synthesis in differentiated and dedifferentiated hepatoma cell lines
Authors: BÖSZE Zsuzsanna , ARÁNYI Péter , VENETIANER Anikó 
Published: March 2005

The pattern of protein synthesis in hepatoma cell clones was analysed by two‐dimensional separation of [35S]methionine‐labelled proteins. The clones were derived from the differentiated Reuber H35 hepatoma and showed differences in the expression of a number of liver‐specific functions and the resistance to the growth‐inhibitory effect of glucocorticoids.Five protein spots were observed in the extracts of the differentiated Faza 967 cells that were absent from the electrophoretogram of the dedifferentiated H56 cells. This clone, on the other hand, displayed six spots absent from Faza 967 cells. The growth of both Faza 967 and H 56 cells was strongly inhibited by 1 γM dexamethasone.The dexamethasone‐resistant clone 2, a dedifferentiated derivative of Faza 967 cells, synthesized two polypeptidies that were not present in Faza 967 or H 56 cells and produced four polypeptides at a lower level than Faza 967 cells. The examination of the short‐term effect of dexamethasone on protein synthesis in Faza 967 cells revealed nine induced and one repressed protein spots, which appeared to be in good agreement with earlier published data. It is concluded that dedifferentiation, although bringing about marked changes in certain liver‐specific functions, such as enzyme activities or protein secretion, affects only a relatively small fraction of the genes expressed.
Aglycon specificity of fetal calf liver and ovine and porcine submaxillary gland α‐N‐acetylgalactosaminide α2→6 sialyltransferase
Authors: BERGH Michel L. E. , VAN DEN EIJNDEN Dirk H. 
Published: March 2005

The specificity of fetal calf liver and ovine and porcine submaxillary gland N‐acetylgalactosaminide α2→6 sialyltransferase was investigated with acceptors of low and high molecular weight containing O‐glycosidically linked carbohydrate chains. It appeared that fetal calf liver microsomes were able to transfer sialic acid to C‐6 of GalNAc in NeuAc(α2→3)Gal(β1→3)GalNAc‐R, in which the aglycon could be protein as well as p‐nitrophenol (Nph). The substrates Gal(β1→3)GalNAc‐R and GalNAc‐R were inactive as acceptor with this enzyme source. Ovine and porcine submaxillary gland microsomes were both active with GalNAc‐protein, Gal(β1→3)GalNAc‐protein, NeuAc(α2→3)Gal(β1→3)GalNAc‐protein and NeuAc(α2→3)Gal(β1→3)GalNAcα‐Nph, but not with GalNAcα‐Nph and Gal(β1→3)GalNAcα‐Nph. The N‐acetylgalactosaminide α2→6 sialyltransferase which had been purified to homogeneity from porcine submaxillary gland [Sadler, J. E., Rearick, J. I. and Hill, R. L. (1979) J. Biol. Chem. 254, 5934–5941], was able to sialylate all three protein acceptors, but was virtually inactive with each of the three p‐nitrophenyl glycosides.Our studies indicate that two N‐acetylgalactosaminide α2→6 sialtransferases exist acting on O‐glycosidically linked carbohydrate chains. The first enzyme, present in fetal calf liver, has a narrow specificity with regard to the oligosaccharide structure, but shows no specificity for the aglycon. Based on its specificity this enzyme can be designated as an [α‐N‐acetylneuraminosyl2→3‐β‐galactosyl 1→3]‐α‐N‐acetylgalactosaminide α2→6 sialyltransferase. The second enzyme, present in porcine submaxillary gland, has an absolute requirement for protein as the aglycon. Once this condition is fulfilled, the enzyme is able to transfer sialic acid to each of the three oligosaccharide chains and thus the enzyme is an α‐N‐acetylgalactosaminylprotein α2→6 sialyltransferase. The data also seem to suggest that ovine and porcine submaxillary gland microsomes contain, in addition to the latter enzyme activity, the α2→6 sialyltransferase with the strict oligosaccharide specificity.
Evidence for an essential histidine residue in glucose dehydrogenase from Bacillus megaterium and sequence analysis of the peptides labeled with bromoacetyl pyridine
Authors: ULMER Wolfgang , FRÖSCHLE Marion , JANY Klaus‐Dieter 
Published: March 2005

Bromoacetylpyridine acts as an active‐site‐directed inhibitor on glucose dehydrogenase from Bacillus megaterium. The inactivation is irreversible with a Ki of 7.7 mM. The coenzyme NAD but not the substrate glucose protects the enzyme from the inactivation. It is proposed that bromoacetylpyridine modifies a residue at or nearby the active site.
The inactivation is correlated with the modification of a single histidine residue.
Modification of the enzyme with 3‐(2‐bromo[carbonyl‐14C]acetyl)‐pyridine and partial acid hydrolysis of the protein yielded one labeled fragment. From the arginine restricted tryptic cleavage of this fragment four radioactively labeled peptides were purified. Comparison of the specific radioactivity leads to the conclusion that the active site histidine residue must be located in the 58‐residue fragment AH2‐TA3. Sequence analysis showed that only one residue is modified in this fragment and the sequence around the labeled histidine residue is ‐Met‐Ser‐Ser‐Val‐His‐Glu‐Trp‐Lys‐Ile‐Pro‐Trp‐Pro‐.
The minor labeled arginine fragments, comprising 86, 20 and 13 residues, were also sequenced. Only lysine residues are modified in these peptides. The modification of the individual residues does not exceed 10%.
Structural studies on the non‐toxic lipid A from Rhodopseudomonas sphaeroides ATCC 17023
Authors: SALIMATH Paramahans V. , WECKESSER Jürgen , STRITTMATTER Wolfgang , MAYER Hubert 
Published: March 2005

The lipid A isolated from Rhodopseudomonas sphaeroides ATCC 17023 has been investigated. A sequence of analyses indicated β‐D‐glucosaminyl‐1,6‐D‐glucosamine as the sugar backbone carrying phosphate groups at C‐1 of the reducing glucosamine and C‐4 of the non‐reducing glucosamine. Both 3‐(7‐tetradecenoyl)oxytetradecanoic and 3‐oxotetradecanoic acid are linked to the NH2 groups. Two residues of 3‐hydroxydecanoic acid are linked to hydroxy groups at C‐3 of both reducing and non‐reducing glucosamine, and the hydroxy group of 3‐hydroxydecanoic acid is free. In free lipid A, the hydroxy group at C‐4 of the reducing glucosamine and at C‐6 of the non‐reducing glucosamine are unsubstituted. The latter probably arises from a 3‐deoxy‐D‐manno‐octulosonate linkage.The structural similarities and dissimilarities existing between lipid A of R. sphaeroides, which is non‐toxic, and lipid A types such as those from Salmonella, which are toxic, are discussed.
A potent new dipeptide inhibitor of cell sickling and haemoglobin S gelation
Authors: FRANKLIN Ian M. , COTTER Rosalind I. , CHEETHAM Rosemary C. , PARDON John F. , HALE Arthur J. , HUEHNS Ernst R. 
Published: March 2005

A dipeptide l‐lysine‐l‐phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sicklecell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys‐Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys‐Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys‐Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3‐bisphosphoglycerate.The effect of Lys‐Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys‐Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane‐mediated anti‐sickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume.The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.
Correlation between calmodulin‐dependent increase in the rate of calcium transport and calmodulin‐dependent phosphorylation of cardiac sarcoplasmic reticulum
Authors: PLANK Brigitte , PIFL Christian , HELLMANN Gertrude , WYSKOVSKY Wolfgang , HOFFMANN Ruth , SUKO Josef 
Published: March 2005

The aim of the present study was to prove a correlation between the calmodulin‐dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin‐dependent phosphorylation.The dependence of phosphorylation on the total calmodulin concentration at 75 μM and 1 μM free calcium gave apparent calmodulin half‐saturation constants Km(CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km(CaM) for the rate of calmodulin‐stimulated calcium transport carried out at 1 μM calcium, but phosphorylated prior to the calcium uptake at 75 μM or 1 μM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin‐dependent increase in the rate of calcium transport and hydroxylamine‐insensitive phosphoester formed by the calcium/calmodulin‐regulated, membrane‐bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26–28‐kDa or 9–11‐kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37°C and at 100°C, respectively, similar to the results obtained by phosphorylation with cAMP‐dependent protein kinase. The data indicate that calmodulin‐dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 μM and 0.61 μM in controls and calmodulin‐dependent phosphorylation, respectively.Activation of calmodulin‐dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 μM, 1.44 μM and 2.3 μM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin‐regulated protein kinase. The calmodulin‐dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.
The primary structure of Escherichia coli glutaredoxin
Authors: HÖÖG Jan‐Olov , JÖRNVALL Hans , HOLMGREN Arne , CARLQUIST Mats , PERSSON Magnus 
Published: March 2005

An immunosorbent method using antiglutaredoxin‐Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10–17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu‐specific extracellular protease.The single active‐center disulfide has the structure , with the half‐cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione–disulfide transhydrogenase).The half‐cystines are identically spaced and similarly positioned in the N‐terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox‐active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms.The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox‐active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide‐adjacent tryptophan residues of E. coli thioredoxin.There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.
Gene fusions using the ompA gene coding for a major outer‐membrane protein of Escherichia coli K12
Authors: HENNING Ulf , COLE Stewart T. , BREMER Erhard , HINDENNACH Ingrid , SCHALLER Heinz 
Published: March 2005

It has been shown previously that fragments of the Escherichia coli major outer membrane protein OmpA lacking CO2H‐terminal parts can be incorporated into this membrane in vivo [Bremer et al. (1982) Eur. J. Biochem. 122, 223–231]. The possibility that these fragments can be used, via gene fusions, as vehicles to transport other proteins to the outer membrane has been investigated.To test whether fragments of a certain size were optimal for this purpose a set of plasmids was prepared encoding 160, 193, 228, 274, and 280 NH2‐terminal amino acids of the 325‐residue OmpA protein. The 160‐residue fragment was not assembled into the outer membrane whereas the others were all incorporated with equal efficiencies. Thus, if any kind of OmpA‐associated stop transfer is required during export the corresponding signal might be present between residues 160 and 193 but not CO2H‐terminal to 193.The ompA gene was fused to the gene (tet) specifying tetracycline resistance and the gene for the major antigen (vpl) of foot‐and‐mouth disease virus. In the former case a 584‐residue chimeric protein is encoded consisting NH2‐terminally of 228 OmpA residues followed by 356 CO2H‐terminal residues of the 396‐residue ‘tetracycline resistance protein’. In the other case the same part of OmpA is followed by 250 CO2H‐terminal residues of the 213‐residue Vp1 plus 107 residues partly derived from another viral protein and from the vector. Full expression of both hybrids proved to be lethal.Lipophilic sequences bordered by basic residues, present in the non‐OmpA parts of both hybrids were considered as candidates for the lethal effect. A plasmid was constructed which codes for 280 OmpA residues followed by a 31‐residue tail containing the sequence: ‐Phe‐Val‐Ile‐Met‐Val‐Ile‐Ala‐Val‐Ser‐Cys‐Lys‐. Expression of this hybrid gene was lethal but by changing the reading frame for the tail to encode another, 30‐residue sequence the deleterious effect was abolished. It is possible that the sequence incriminated acts as a stop signal for transfer through the plasma membrane thereby jamming export sites for other proteins and causing lethality. If so, OmpA appears to cross the plasma membrane completely during export.
Interaction of mature, unglycosylated and cell‐free synthesized rat α1‐proteinase inhibitor with elastase
Authors: ANDUS Tilo , GROSS Volker , TRAN‐THI Thuy‐Anh , HEINRICH Peter C. 
Published: March 2005

Purified rat α1‐proteinase inhibitor (Mr 54 000) and porcine pancreatic elastase (Mr 26 000) formed a complex (Mr 82 000) resistant to heat treatment under denaturing and reducing conditions. A similar α1‐proteinase‐inhibitor‐elastase complex was detected by immunoprecipitation of α1‐proteinase inhibitor from the medium of [35S]methionine‐labeled hepatocyte primary cultures after incubation with elastase. Treatment of hepatocytes with tunicamycin led to the secretion of an unglycosylated α1‐proteinase inhibitor (Mr 41 000) which also formed a complex with elastase (Mr 66 000). Complex formation (Mr 68 000) could also be observed between cell‐free synthesized pre‐α1‐proteinase inhibitor (Mr 43 000) and elastase (Mr 26 000).The results suggest that neither glycosylation nor removal of the signal peptide are required for the formation of a biologically active conformation of α1‐proteinase inhibitor.
Intracellular forms of transferrin oligosaccharide chains in rat liver
Authors: NAKADA Hiroshi , KOHNO Hirao , KAWASAKI Toshisuke , TASHIRO Yutaka 
Published: March 2005

Glycosylation of transferrin was investigated in vivo by using antibody monospecific for rat serum transferrin. Pulse‐chase experiments indicated that, after intravenous injection of [35S]methionine, labeled transferrin appeared in the rough and smooth microsomes and Golgi subfractions in rapid succession in 10 min and that an additional 10 min was required for it to be secreted.Most of the intracellular transferrin (95%) immunoprecipitated from the total microsome fraction was sensitive to endo‐β‐N‐acetylglucosaminidase H (endo H), whereas serum transferrin was completely resistant to it. Further fractionation of the total microsomes has revealed that the intracellular transferrin immunoprecipitated from the rough and smooth microsomes and GF3 are all endo‐H‐sensitive and most of the endo‐H‐sensitive oligosaccharides were eluted at the position corresponding to Man8GlcNAc on high‐resolution Bio‐Gel chromatography. This finding suggests that the major form of intracellular transferrin oligosaccharide in the course of intracellular transport from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2.Endo‐H‐resistant forms were first detected in the GF2 but more in GF1, most of which were sensitive to neuraminidase. Since the heavy Golgi subfraction contains mainly cis‐Golgi elements, such as cisternae, and the light subfraction mainly trans‐Golgi elements, such as secretory granules, it is strongly suggested that the processing of these large mannosyloligosaccharide chains and the subsequent addition of terminal sugars to them are performed successively in the trans‐Golgi region just before secretion.
Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria
Authors: TOMASSELLI Alfredo G. , DOUGHERTY John J. , NODA Lafayette H. 
Published: March 2005

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26000; MgGTP + AMP ⇆ MgGDP + ADP) has been attained by treatment of the enzyme with N‐bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (λexc 295 nm) were recorded over the pH range 1.9–12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M−1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0–5.5 the fluorescence peak has a constant height with maximum at 333–334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N‐bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride.In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the λmax of emission shifts from 333–334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non‐radiative energy transfer to tyrosinate.
Stereochemistry of the rearrangement of 2‐aminoethanol by ethanolamine ammonia‐lyase
Authors: GANI David , WALLIS O. Caryl , YOUNG Douglas W. 
Published: March 2005

(1R)‐2‐Amino[1‐2H1]ethanol and (1S,2RS)‐2‐amino[1,2‐2H2]ethanols have been synthesised by decarboxylation of (2S,3R)‐[3‐2H1]serine and (2S,3S)‐[2,3‐2H2]serine respectively.
The stereochemical integrity of these labelled 2‐aminoethanols has been ascertained from the 1H‐NMR spectra of their N,O‐dicamphanoyl derivatives.
This assay has also been used to confirm that samples of (2R)‐ and (2S)‐2‐amino[2‐2H1]ethanols prepared from (2R)‐ and (2S)‐[2‐2H1]glycines are stereochemically pure.
Ethanolamine ammonia‐lyase rearranges (1R)‐2‐amino[1‐2H1]ethanol to acetaldehyde at approximately the same rate as it rearranges unlabelled 2‐aminoethanol, whilst (1S,2RS)‐2‐amino[1,2‐2H2]ethanol is rearranged at the same rate as the (1,1‐2H2)‐labelled substrate. The isotope effect is approximately kH/k2H= 8.
The 2H‐NMR spectra of the 3,5‐dinitrobenzoates of the ethanol produced by reduction in situ of the acetaldehyde formed in the rearrangements show that the 1‐2H1 label migrates in (1S,2RS)‐2‐amino‐[1,2‐2H2]ethanol and 2‐amino[1,1‐2H2]ethanol but not in (1R)‐2‐amino[1‐2H1]ethanol.
The above results indicate that the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase catalyses the rearrangement of 2‐aminoethanol with migration of the 1‐pro‐S‐hydrogen atom.