bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of a vasodilator‐regulated phosphoprotein from human platelets
Authors: HALBRÜGGE Maria , WALTER Ulrich 
Published: March 2005

Cyclic‐nucleotide‐elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium‐derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade.Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441–448) established that in human platelets cyclic‐nucleotide‐elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP‐ and cGMP‐dependent protein kinases. Of particular interest was a membrane‐bound 50‐kDa protein whose phosphorylation was increased both by cAMP‐and cGMP‐elevating vasodilators in intact platelets and by endogenous cAMP‐ and cGMP‐dependent protein kinase in platelet membranes.Since the molecular mechanism of action of cyclic‐nucleotide‐elevating vasodilators is unknown, this 50‐kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye‐ligand chromatography. The purified protein migrated as a 46‐kDa protein in SDS/PAGE, was an excellent substrate for both cAMP‐ and cGMP‐dependent protein kinases and migrated in SDS/PAGE as a 50‐kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50‐kDa protein phosphorylated by both cAMP‐ and cGMP‐dependent protein kinases in platelet membranes and in response to cAMP‐ and cGMP‐elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP‐ or cGMP‐dependent protein kinase. The availability of this vasodilator‐regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic‐nucleotide‐elevating vasodilators.
Gangliosides of sea urchin embryos
Authors: ZVEZDINA Natalia D. , SADYKOVA Kamila A. , MARTYNOVA Larisa E. , PROKAZOVA Nina V. , MIKHAILOV Alexander T. , BUZNIKOV Gennady A. , BERGELSON Lev D. 
Published: March 2005

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. γ‐Globulins were isolated from rabbit anti‐ganglioside serum by micropreparative electrophoresis. These γ‐globulins produced anomalies in the development of embryos permeabilized in Triton X‐100. The anomalies were not observed when anti‐ganglioside γ‐globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with γ‐globulins of normal rabbit serum.Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell‐associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl‐Sepharose.These observations suggest that cell‐surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell‐surface membrane.
Pregnenolone synthesis from cholesterol and hydroxycholesterols by mitochondria from ovaries following the stimulation of immature rats with pregnant mare's serum gonadotropin and human choriogonadotropin
Authors: TUCKEY Robert C. , ATKINSON Helen C. 
Published: March 2005

The rate of pregnenolone synthesis by cytochrome P‐450scc was measured in mitochondria isolated from ovaries of immature rats treated with pregnant mare's serum gonadotropin and human choriogonadotropin. Using cholesterol, 25‐hydroxycholesterol, 20α‐hydroxycholesterol, (22R)‐22‐hydroxycholesterol and (22R)‐20α,22‐dihydroxycholesterol as substrates, we have determined that the first hydroxylation of cholesterol, in the 22R position, is rate limiting in pregnenolone synthesis. It proceeds at only 22% of the rate of either of the subsequent two hydroxylations.25‐Hydroxycholesterol proved to be a suitable substrate for determining the maximum rate of pregnenolone synthesis by cytochrome P‐450scc in isolated mitochondria. The maximum rate was 13 mol steroid · min−1· mol cytochrome P‐450−1scc and did not change after the follicles in the immature ovary had been stimulated to mature and luteinize with gonadotropin.Using endogenous cholesterol in isolated mitochondria as substrate, the time course of pregnenolone synthesis was the same during the follicular phase as in the luteal stage of gonadotropin‐induced development. We conclude that during the artificially induced development of follicles in the immature ovary, the major cause of the increase in the rate of pregnenolone synthesis is the increase in the cytochrome P‐450scc content of the mitochondria, rather than changes in the catalytic activity of cytochrome P‐450scc or the cholesterol availability to the cytochrome.
Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells
Authors: PFEIFFER Günter , SCHMIDT Martin , STRUBE Karl‐Hermann , GEYER Rudolf 
Published: March 2005

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6‐3H]‐glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H‐NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N‐terminal amino acid sequencing.The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5–8 mannose residues as well as small amounts of hybrid‐type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N‐acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(α1–3) substituents and/or sulfate groups linked to position six of β‐galactosyl residues forming NeuAc(α2–3)[HO3S–6]Gal(β1–4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (α2–3)‐ or (α2–6)‐linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.
13C and 15N NMR and time‐resolved fluorescence depolarization study of bovine carbonic anhydrase—4‐methylbenzenesulfonamide complex
Authors: JARVET Jüri , OLIVSON Abira , METS Ülo , POOGA Margus , AGURAIUJA Reet , LIPPMAA Endel 
Published: March 2005

The influence of the binding of the high‐affinity inhibitor, 4‐methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N‐ and 13C‐NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time‐resolved fluorescence depolarization measurements.Our experiment provides evidence that the stoichiometry of the interaction of 4‐methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N‐NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme‐inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.
Protein–protein interactions in microsomal cytochrome P‐450 isozyme LM2 and their effect on substrate binding
Authors: HILDEBRANDT Peter , GARDA Horacio , STIER Anton , BACHMANOVA Galina I. , KANAEVA Irina P. , ARCHAKOV Alexander I. 
Published: March 2005

The effects of protein–protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P‐450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2‐dependent catalytic activities of the two states have been compared.The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P‐450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P‐450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin‐state equilibrium. This indicates that in the oligomer the substrate‐binding pocket is converted by protein–protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five‐coordinated high‐spin configuration. In the monomer the substrate‐binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P‐450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein–protein interactions.H2O2‐dependent N‐demethylation of benzphetamine and aniline p‐hydroxylation by cytochrome P‐450 LM2 did not depend on its state of aggregation.
Crosslinking of cytochrome c and cytochrome b5 with a water‐soluble carbodiimide
Authors: MAUK Marcia R. , MAUK A. Grant 
Published: March 2005

A water soluble carbodiimide, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), has been used to crosslink horse heart cytochrome c and trypsin‐solubilized bovine liver microsomal cytochrome b5. The reaction was conducted under a variety of solution conditions, and the products were purified by a combination of gel filtration and ion‐exchange chromatography. Under all conditions of pH, ionic strength, EDC/protein ratio and reaction time that were studied, multiple 1:1 crosslinked complexes were observed with no evidence of a single, dominant species. Acetate, which is often used as a quencher of such reactions, was found to increase the complexity of the reaction products, presumably through EDC‐promoted coupling to cytochrome c. Hydroxylamine treatment of the crosslinked complexes, a procedure frequently used to reverse EDC modification of tyrosyl residues, did not reduce the number of crosslinked components observed. The cytochrome b5 heme group was readily extracted from each of the 1:1 crosslinked complexes by standard techniques, so the crosslinking of heme propionate 7 with Lys79 of cytochrome c that might have been anticipated on the basis of molecular graphics modeling [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568] was not evident from this analysis.Analysis of HPLC tryptic peptide maps produced from crosslinked complexes revealed reduced specificity of trypsin in hydrolysis of EDC‐crosslinked protein‐protein complexes and unsatisfactory resolution of crosslinked or branched peptides. Nevertheless, it was possible to demonstrate that residues 52–72 of cytochrome b5, a region predicted to be critical to interaction with cytochrome b5 [Salemme, F. R. (1976) J. Mol. Biol. 102, 563–568] was absent from all peptide maps of 1:1 cytochrome c· cytochrome b5 complexes. Based on these results and a review of the literature involving EDC crosslinking of electron transfer proteins, we conclude that the techniques available for specific protein hydrolysis and separation of crosslinked peptides are not adequate to permit routine unambiguous identification of crosslinking sites in carbodiimide‐crosslinked complexes.
O‐specific polysaccharides of Hafnia alvei lipopolysaccharides isolated from two serologically related strains: ATCC 13337 and 1187
Authors: GAMIAN Andrzej , ROMANOWSKA Elzbieta , OPFERKUCH Hans J. , HAUCK Michael , DABROWSKI Janusz 
Published: March 2005

The O‐specific polysaccharides of Hafnia alvei ATCC 13337 standard strain and 1187 strain have been isolated and characterized. By means of 1H‐NMR spectroscopy, methylation analysis and periodate oxidation, the repeating unit of the polysaccharides could be allocated the respective structures:

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where Acyl = D‐3‐hydroxybutyryl, and 3‐O‐acetylation was to about 66%.The structural similarity of the polysaccharides was confirmed in the serological study: their epitopes were determined and the importance of various structural elements for the serological specificity was discussed.
Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor
Authors: RALTON Julie E. , JACKSON Heather J. , ZANONI Maureen , GLEESON Paul A. 
Published: March 2005

The murine transferrin receptor is a disulphide‐linked dimer with three N‐glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS‐1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation‐processing inhibitors, swainsonine and castanospermine.

Tunicamycin (1 μg/ml) inhibited mannose incorporation in NS‐1 cells by > 90%, but also inhibited me‐thionine incorporation by up to 50%. Both swainsonine (1 μg/ml) and castanospermine (50 μg/ml) resulted in mannose incorporation > 100% of untreated cells and neither drug affected methionine incorporation.
Incubation of NS‐1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa.
Peptide N‐glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N‐glycosidase F digestion.
The receptor from swainsonine‐treated cells was equally sensitive to peptide N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase H (endo H; resulting in both 87‐kDa and 82‐kDa components), whereas the receptor from castanospermine‐treated cells was only partially sensitive to endo H.
Analysis of mannose‐ and fucose‐labelled cellular glycopeptides by concanavalin‐A ‐ Sepharose chromatography showed that swainsonine (1 μg/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N‐glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 μg/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N‐glycans.
Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under non‐reducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesisted murine receptor is independent of carbohydrate.
The non‐glycosylated receptor from tunicamycin‐treated cells appears to bind transferrin as demonstrated by interaction with transferrin‐Sepharose.
Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.
Distribution analyses of chain substituents of lipoteichoic acids by chemical degradation
Authors: SCHUREK Josef , FISCHER Werner 
Published: March 2005

The lipoteichoic acid fromLactococcus lactisKiel 48337 was analyzed. It had 61% of its glycerophosphate residues substituted with α‐d‐galactopyranosyl residues. Non‐substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage. The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE‐Sephadex into 10 pairs of molecular species withnfrom 1 to 10. The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents. A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid. Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern.The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P= 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.
Structural homology between glycophorins C and D of human erythrocytes
Authors: EL‐MALIKI Bouchra , BLANCHARD Dominique , DAHR Wolfgang , BEYREUTHER Konrad , CARTRON Jean‐Pierre 
Published: March 2005

Glycophorin C (GPC) and D (GPD) are minor glycoproteins which are believed to be important for the structural integrity of the red cell membrane.We have investigated the structural relationship between these glycoproteins by both immunological and structural investigations:

A rabbit anti‐serum produced against GPD reacts strongly with GPC and the abnormal glycoproteins of Gerbich: –2, –3 and Gerbich: –2,3 red cells, and recognizes most probably the homologous C‐terminal portions of GPC and GPD. The two molecules however differ at their N‐terminus.
One‐dimensional mapping of the peptides obtained after tryptic, chymotryptic, V8 protease or acid cleavage of 125 I‐labelled GPC and GPD, indicated that GPC and GPD are structurally related but some differences were found indicating that additional peptides were generated from GPC.
The partial primary structure of GPD was determined. The sequencing data are consistent with the assumption that GPD represents an abridged version of GPC that comprises residues approximately 21/29–128 and exhibits a N‐terminal residue that is blocked by an as yet undefined group.
Novel inhibitors and substrates of bilirubin: UDP‐glucuronosyltransferase Arylalkylcarboxylic acids
Authors: FOURNEL‐GIGLEUX Sylvie , SHEPHERD Steven R. P. , CARRE Marie‐Christiane , BURCHELL Brian , SIEST Gérard , CAUBERE Paul 
Published: March 2005

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP‐glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7‐triphenylheptanoic acid was the most powerful inhibitor of bilirubin: UDP‐glucuronosyltransferase with a lower effect on 1‐naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin: UDP‐glucuronosyltransferases with Kiapp values of 12.0 μM and 1.6μM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin: UDP‐glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group.These inhibitors were also tested as possible substrates of UDP‐glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP‐glucuronosyltransferases. However, 7,7,7‐triphenylheptanoic acid was actively glucuronidated by purified bilirubin: UDP‐glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin: UDP‐glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin: UDP‐glucuronosyltransferase.The results suggest that 7,7,7‐triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.
Identification and characterization of the major chicken bone phosphoprotein
Authors: GOTOH Yozo , GERSTENFELD Louis C. , GLIMCHER Melvin J. 
Published: March 2005

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O‐[32P]phosphoserine and O‐[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ∼ 66‐kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ∼ 66‐kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE‐Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino‐terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine‐residue sequence spanning the Arg‐Gly‐Asp cell‐binding site of the rat protein osteopontin, was identified in the ∼ 66‐kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions.Several smaller‐molecular‐mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HC1 extracts of bone. Peptide mapping of these species demonstrated that the ∼ 66‐kDa, ∼ 55‐kDa, and ∼ 45‐kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ∼ 66‐kDa species since this phosphoprotein remained the prevalent species after a 24‐h chase. Because of the predominant association of all the smaller‐molecular‐mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24‐h chase, these results suggested that the lower‐molecular‐mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ∼ 66‐kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major ∼ 66‐kDa phosphoprotein that is identical to the major HCl‐extractable phosphoprotein present in postnatal chick bone.