bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational transitions within the head and at the head‐rod junction in smooth muscle myosin studied with a limited proteolysis method
Authors: RĘDOWICZ Maria J. , SOBIESZEK Apolinary , STRZELECKA‐GOŁASZEWSKA Hanna 
Published: March 2005

It was previously shown that tryptic digestion of subfragment 1 (S1) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH2‐terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25°C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH2‐terminus is involved in the communication between the sites of nucleotide and actin binding.We also show here for the first time that the S1–S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain‐produced S1 devoid of the regulatory light chain) is also temperature‐dependent but insensitive to nucleotides and actin. It is suggested that the temperature‐dependent alteration in the flexibility of the head‐rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J. Mol. Biol. 182, 271–279; Rȩdowicz, M. J. & Strzelecka‐Golaszewska, H. (1988). Eur. J. Biochem. 177, 615–624], may contribute to the temperature dependence of some steps in the cross‐bridge cycle.
Primary structure of the oligo‐1,6‐glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene
Authors: WATANABE Kunihiko , KITAMURA Kazuhisa , IHA Hidekatsu , SUZUKI Yuzuru 
Published: March 2005

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo‐1,6‐glucosidase was cloned within a 2.8‐kb SalI—EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo‐1,6‐glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo‐1,6‐glucosidase in its Mr (65000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants.The nucleotide sequence of B. cereus oligo‐1,6‐glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66010. The amino acid composition and Mr were comparable with those of B. cereus oligo‐1,6‐glucosidase. The predicted N‐terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo‐1,6‐glucosidase.The deduced amino acid sequence of B. cereus oligo‐1,6‐glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo‐1,6‐glucosidase and from Saccharomyces carlsbergensis CB11 α‐glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo‐1,6‐glucosidase may take the similar (α/β)8‐barrel super‐secondary structure, a barrel of eight parallel β‐strands surrounded by eight α‐helices, in its N‐terminal active site domain as S. carlsbergensisα‐glucosidase and Aspergillus oryzaeα‐amylase.
Site‐specific N‐glycosylation of ovine lutropin
Authors: WEISSHAAR Gerhard , HIYAMA Jun , RENWICK Alistair G. C. 
Published: March 2005

The Asn‐linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one‐ and two‐dimensional 400‐MHz 1H‐NMR spectroscopy.Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its α and β subunits (oLHα, glycosylated at Asn56 and Asn82; oLHβ glycosylated at Asn13). Oligosaccharides from intact oLHβ and from glycopeptides obtained after tryptic digestion of oLHα were released by hydrazinolysis and subsequently fractionated according to charge and size by anion‐exchange and ion‐suppression amine‐adsorption HPLC, respectively. 1H‐NMR analysis revealed, that monosulphated, mostly hybrid‐type, oligosaccharides predominate at both glycosylation sites of oLHα, whereas a disulphated, diantennary N‐acetyllactosamine‐type structure accounts for more than 60% of total oligosaccharides in the β subunit. Furthermore, the saccharides attached to the β subunit are almost completely fucosylated (Fucα1–6) at the reducing terminal GlcNAc, whereas the sugar chains in oLHα are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56).The results clearly indicate a distinct subunit‐ and site‐specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.
Primary structure of profilins from two species of Echinoidea and Physarum polycephalum
Authors: TAKAGI Takashi , MABUCHI Issei , HOSOYA Hiroshi , FURUHASHI Kiyoshi , HATANO Sadashi 
Published: March 2005

Profilin is a small G‐actin‐binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N‐termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465–476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N‐terminus was acylated, and the calculated molecular mass was 13132 Da.The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those from other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins.The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N‐terminal region, which was suggested to be a common actin‐binding region. The C‐terminal region was also conserved, although to a lesser extent than the N‐terminal region.
Characterization of the transcription‐initiation site and of the promoter region within the 5′ flanking region of the human aldolase C gene
Authors: BUONO Pasqualina , MANCINI Francesco P. , IZZO Paola , SALVATORE Francesco 
Published: March 2005

Several aldolase C clones from a human genomic library have been identified using a mouse aldolase C cDNA as a hybridization probe. The most complete fragment of the clones identified is 14 kb long and contains the complete aldolase C gene. The nucleotide sequence analysis of more than 5 kb includes the intron/exon organization structure of the gene and the 3′ and 5′ flanking regions. Although no human cDNA is yet available, a canonical polyadenylation signal at the 3′ end of the gene indicates the proximity of the poly(A) addition site.We have analyzed the 5′ noncoding region by S1 mapping and primer‐extension experiments. The transcription‐initiation site for the human aldolase C gene in brain tissue was located about 1300 bp upstream from the methionine initiation codon.Preliminary functional assays of the promoter by transfection into rat glioma cells have indicated that promoter elements lie between positions −161 and −416 from the start point of transcription.
Secretin receptors in the neuroglioma hybrid cell line NG108‐15
Authors: GOSSEN Denis , TASTENOY Michèle , ROBBERECHT Patrick , CHRISTOPHE Jean 
Published: March 2005

Secretin receptors in membranes from the neuroblastoma‐glioma hybrid cell line NG108‐15 were investigated by 125I‐secretin binding and adenylate cyclase activation. On both parameters the corresponding relative potencies of parent peptides were, respectively: secretin > helodermin > peptide histidine isoleucinamide = vasoactive intestinal peptide. With secretin analogs and secretin fragments, the order of potency for binding was: secretin = [Val5]secretin > [Ala2]secretin = [Ala11]secretin > [Ala4,Val5]secretin > [Ala4]secretin > [d‐Phe4]secretin > [d‐Phe2]secretin = secretin (2–27) > secretin (3–27) > secretin (7–27). Also, on adenylate cyclase, [d‐Phe4]secretin, [d‐Phe2]secretin, secretin (2–27) and secretin (3–27) were partial agonists while secretin (7–27) was ineffective.The differentiating agent N6,2′‐O‐dibutyryladenosine 3′,5′‐monophosphate (1 mM) increased the density of secretin receptors and secretin‐stimulated adenylate cyclase activity after a lag period of 4 h. After incubation for 24 h, receptor number and enzyme activity were increased 4‐ and 3‐fold, respectively. These effects were inhibited totally by 1 μg/ml cycloheximide and halved by 5 μg/ml actinomycin D. They were mimicked by 1 mM sodium butyrate but were not reproduced by either 8‐bromoadenosine 3′,5′‐monophosphate or the phosphodiesterase inhibitor rac‐4‐(3‐Butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone.
Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na+‐exchange reaction of the sodium pump unaltered and shifts its high‐affinity ATP‐binding site to a Na+‐like form
Authors: BUXBAUM Engelbert , SCHONER Wilhelm 
Published: March 2005

Inactivation of Na+/K+‐ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb+/Rb+ exchange, but leaves the 22Na+/Na+‐exchange activity and the uncoupled ATP‐supported 22Na+ transport unaffected.Furthermore, inactivation of purified Na+/K+‐ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi‐supported pathway (ouabain‐enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP‐supported pathway (ouabain‐enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K+‐ATPase.When eosin was used as a marker for the high‐affinity ATP‐binding site of the E1 conformation, formation of stable E′2· Co(NH3)4PO4 complex led to a shift in the high‐affinity ATP‐binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4‐inactivated enzyme.It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an α,β‐protomeric enzyme working according the Albers‐Post scheme. The data are consistent with an α2,β2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers‐Post model.
myo‐Inositol oxygenase from rat kidneys
Authors: KOLLER Franz , KOLLER Elisabeth 
Published: March 2005

myo‐Inositol from rat kidneys, an oligomeric protein with apparent molecular mass of about 270 kDa can be dissociated under mild conditions to structured 16.8‐kDa monomers. This dissociation can be reversed at high protein concentrations at room temperature. The corresponding apparent dimerization constant K2app = 1.38 × 105 M−1, the corresponding rate constant k2= 350 s−1· M−1, and the apparent constant for the association of dimers, K4app = 2.7 × 106 M−1.Reassociation is significantly enhanced in the presence of the substrate and iron(II) (K2app= 9.8 × 105 M−1; K4app= 3.75 × 106 M−1, k2= 1750 s−1· M−1, at 20 mM myo‐inositol and 0.5 mM FeSO4). Under these conditions almost 100% of the original enzymatic activity was reconstituted.Monomers, with or without bound ligands, lack catalytic activity, whereas the dimer is likely to be the elementary active enzyme‐building unit. The effects of myo‐inositol on the dimerization lead to the conclusion that this step is both mediated and facilitated by the substrate.
Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells
Authors: GEYER Hildegard , KEMPF Roland , SCHOTT Hans‐Henning , GEYER Rudolf 
Published: March 2005

A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus‐inducing virus plus the remainder of the genome of an amphoropic murineleukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2‐3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo‐ß‐N‐acetylglucosaminidase H and peptide‐N4‐(N‐acetyl‐ß‐glucosaminyl)asparagine amidase F and fractionated by high‐performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing.The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4‐ and 2,6‐branched triantennary and tetraantennary complex type N‐glycans with 0–4 sialic acid residues and only small amounts of high‐mannose type species with 5–9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(α1–3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4‐branched triantennary oligosaccharides with 1–3 sialic acid residues, in addition to trace amounts of high‐mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N‐glycans of the retroviral envelope glycoprotein display cell‐specific variations including differences in oligosaccharide branching, sialylation and subtitution by additional Gal(α1–3) residues.
On the limited ability of superoxide to release iron from ferritin
Authors: BOLANN Bjørn J. , ULVIK Rune J. 
Published: March 2005

Reductive release of iron from ferritin may catalyze cytotoxic radical reactions like the Haber‐Weiss reaction. The ability of ·O−2 to mobilize Fe(II) from ferritin was studied by using the xanthine/xanthine oxidase reaction, with and without superoxide dismutase, and with bathophenanthroline sulphonate as the chelator. Not more than one or two Fe(II)/ferritin molecules could be released by an ·O−2‐dependent mechanism, even after repeated exposures of ferritin to bursts of ·O−2.The amount of releaseable iron depended on the size and the age of the iron core, but not on the iron content of the protein shell of ferritin which was manipulated by chelators and addition of FeCl3. The kinetic characteristics of the ·O−2 mediated iron release indicated the presence of a small pool of readily available iron at the surface of the core. The very limited ·O−2 dependent release of iron from ferritin is compatible with a protective role of ferritin against toxic iron‐catalyzed reactions.
Interferon‐mediated transcriptional and post‐transcriptional modulation of complement gene expression in human monocytes
Authors: LAPPIN David F. , BIRNIE George D. , WHALEY Keith 
Published: March 2005

The addition of lymphoblastoid interferon α, fibroblast interferon β and recombinant interferon γ to in vitro monocyte cultures produced dose‐dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon γ was the most effective at stimulating transcription of the C1‐inhibitor gene whereas interferons α and β were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon γ. None of these cytokines altered the level of transcription of the actin gene.Interferon‐induced changes in the levels of transcription of the C2, B and C1‐inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines.Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1‐inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1‐inhibitor mRNA remained elevated in interferon‐γ‐treated monocytes.Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1‐inhibitor genes, whereas measurements of C1‐inhibitor mRNA and B mRNA showed that interferon γ acted synergistically with interferon γ to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive.The half‐lives of C1‐inhibitor, C2, B and C3 mRNA were not altered by interferon α, whereas interferon γ shortened the half‐life of C2 mRNA by approximately 50%, and prolonged the half‐lives of B and C1‐inhibitor mRNA approximately twofold and fivefold, respectively. The half‐lives of C3 mRNA was unaltered by either interferon.These results show that the large increase in C1‐inhibitor synthesis which occurs in interferon‐γ‐treated monocytes, is due to a combination of increased transcription and increased C1‐inhibitor mRNA stability. They also suggest that the synergistic effects of interferon α together with interferon γ on C1‐inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.
Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates
Authors: DONELLA‐DEANA Arianna , BRUNATI Anna Maria , MARCHIORI Fernando , BORIN Gianfranco , MARIN Oriano , PINNA Lorenzo A. 
Published: March 2005

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] and conventionally termed TPK‐I, TPK‐IIB and TPK‐III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N‐terminal acidic residue(s) are only essential with TPK‐IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 μM) but 14‐fold‐different Vmax values with YEEEEE vs. EEEEEY indicate that an N‐terminal rather than C‐terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK‐IIB.Acidic residues decrease the phosphorylation rate with TPK‐I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK‐III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK‐I and TPK‐IIB.While the peptide substrate specificity of TPK‐I is similar to that of TPK‐IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145–149], the remarkable requirement of TPK‐IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.
A new force‐field program for the calculation of glycopeptides and its application to a heptacosapeptide‐decasaccharide of immunoglobulin G1
Authors: STUIKE‐PRILL Rainer , MEYER Bernd 
Published: March 2005

Energetically favored conformations of glycopeptide 1 were calculated using the newly developed force‐field program, GEGOP (geometry of glycopeptides). The three‐dimensional structure of glycopeptide 1, which is part of the Fc fragment of IgG1, has been calculated. 1 contains 27 amino acid residues from Pro291 to Lys317 and a biantennary decasaccharide N‐linked to Asn297. The conformations of the peptide and the carbohydrate parts are shown to be mutually dependent. Single glycosyl residues of 1 exhibit interaction energies of up to –31.8 kJ/mol with the peptide portion. Generally, only a few of the glycosyl residues of the oligosaccharide moiety express significant interaction energies with the peptide part. No easy prediction is possible of glycosyl residues which exhibit favorable interaction energies. However, in all of the calculated structures, the glycosyl residues of the 1–6‐linked branches show strong attractive forces for the peptide part. 1–6‐glycosidically linked branches can adopt a larger number of conformations than other linkages due to their high flexibility which allows more favorable interactions with proteins.We developed the GEGOP program in order to be able to study the preferred conformations of large glycopeptides. The program is based on the GESA (geometry of saccharides) program and utilizes the HSEA (hard sphere exo anomeric) force field for the carbohydrate part and the ECEPP/2 (empirical conformation energy program for peptides) force field [Némethy, G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 87, 1883–1887] for the peptide part. The GEGOP program allows the simultaneous relaxation of all rotational degrees of freedom of these glycoconjugates during the energy optimization process. Thus, mutual interactions between glycosyl and amino acid residues can be studied in detail.