bluuebunny/crossref_metadata_2025 huggingface dataset:
The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase
Authors: Pettigrew G. W. , Gilmour R. , Goodhew C. F. , Hunter D. J. B. , Devreese B. , Van Beeumen J. , Costa C. , Prazeres S. , Krippahl L. , Palma P. N. , Moura I. , Moura J. J. G.
Published: March 2003

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X‐ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular‐exclusion chromatography is indicative of an ionic strength‐dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H‐NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross‐linking studies, which showed that a cross‐linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular‐exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H‐NMR spectroscopy shows that it is the monomer that binds.
Identification of highly fucosylated N‐linked oligosaccharides from the human parotid gland
Authors: Guile Geoffrey R. , Harvey David J. , O'Donnell Niall , Powell Andrew K. , Hunter Ann P. , Zamze Susanne , Fernandes Daryl L. , Dwek Raymond A. , Wing David R.
Published: March 2003

The glycosylation of a number of constituents of human saliva is known to modify its biological roles, such as its lubricating properties and binding of microbial flora. Gillece‐Castro et al. [Gillece‐Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17 358−17 368] have proposed that the major glycan on the salivary proline‐rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non‐fucosylated antenna mediated adherence to a peridontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co‐exist in the parotid gland are not fully known. In view of the influence of outer‐arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix‐assisted laser‐desorption/ionisation (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed‐phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal‐phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2‐aminobenzamide‐labelled arabinose oligomers instead of the corresponding oligomers from partially hydrolysed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were additionally found substituted with sialic acid (both α2−3‐linked and α2−6‐linked) and sulphate. These glycans were mainly bi‐ and tri‐antennary sugars with up to five and seven fucose residues respectively, containing fucose α1−3‐linked to the outer‐arm GlcNAc residues and α1−2‐linked to the galactose. All fucosylated structures contained a core (α1−6‐linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amounts of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addition of extra sialic acid moieties. The possible functional significance of highly fucosylated N‐glycans is discussed in relation to their modification of the availability of other non‐reducing terminal monosaccharides for recognition processes. parotid gland; oligosaccharide; matrix‐assisted laser‐desorption/ionization MS; HPLC; fucose.
Structural characterisation of a rhamnan and a fucorhamnan, both present in the lipopolysaccharide of Burkholderia vietnamiensis strain LMG 10926
Authors: Gaur Deepa , Galbraith Lesley , Wilkinson Stephen G.
Published: March 2003

The polymeric fraction isolated after mild acid hydrolysis of the lipopolysaccharide (LPS) from Burkholderia vietnamiensis strain LMG 10926 contained
L‐rhamnose (Rha) and
D‐fucose (Fuc). From NMR studies supported by the results of methylation analysis and Smith degradation, it could be inferred that the material was probably a mixture of two glycans. One component was a linear rhamnan with a trisaccharide repeating unit (1); the other was a branched fucorhamnan with a tetrasaccharide repeating unit (2). The presence of two distinct polymeric fractions in LPS is a common feature for Burkholderia species.

←4)‐α‐ L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐β‐
L‐Rhap‐(1←  

1  

α‐ L‐Rhap

1

↓

2

←3)‐α‐ D‐Fucp‐(1←3)‐α‐
D‐Fucp‐(1←3)‐α‐
L‐Rhap‐(1←  

2
Cloning and sequence analysis of cDNAs encoding human placental tissue protein 17 (PP17) variants
Authors: Than Nandor G. , Sumegi Balazs , Than Gabor N. , Kispal Gyula , Bohn Hans
Published: March 2003

Using monospecific anti‐PP17 serum with chemiluminescence Western‐blot analysis, we detected different molecular‐mass variants of human soluble placental tissue protein 17 (PP17) in different normal adult and fetal human tissues besides term placenta. 13 cDNAs with three different insert lengths encoding PP17 variants were isolated by screening a human placental cDNA library. Sequence analysis of the shortest clones showed that the inserts contain the same open reading frame encoding PP17a variant (28 129 kDa) consisting of 251 residues, which is identical to the previously isolated and characterised PP17 antigen described in 1983. The ubiquitous PP17b variant is encoded by longer clones and contains 434 residues with a predicted molecular mass of 47 208 kDa. Compared to normal conditions, these newly discovered PP17 variants are overexpressed in cervix carcinoma tissue, as are their three different‐size messenger RNAs in HeLa cell line. Increased amounts of PP17b are secreted into the circulation in cervix carcinoma patients. We also observed a typical elevation in serum levels of PP17 variants during healthy pregnancy. An alignment search of the protein databank showed that PP17a and PP17b are homologous to adipose tissue differentiation and lipid‐droplet‐associated proteins : human adipophilin, mouse adipose differentiation‐related protein and rat perilipin A and B.
The sulphoxidation of thioanisole catalysed by lactoperoxidase and Coprinus cinereus peroxidase: Evidence for an oxygen‐rebound mechanism
Authors: Tuynman Antonin , Vink Mandy K. S. , Dekker Henk L. , Schoemaker Hans E. , Wever Ron
Published: March 2003

Using both stopped‐flow and conventional spectroscopy, the oxygenation of methyl phenyl sulphide by both lactoperoxidase (LPO) and Coprinus cinereus peroxidase (CiP) was monitored. Controlled continuous addition of H2O2 during turnover and monitoring the presence of native enzymes, compounds I, II and III, led to formation of the sulphoxide in high yield and enantioselectivity. Under those conditions, LPO catalysed the formation of (R) methyl phenyl sulphoxide with a yield of 85 % and an enantiomeric excess (e.e.) of 80 %. CiP catalysed the formation of (S) methyl phenyl sulphoxide with a yield of 84 % and an e.e. of 73 %. The enantioselective performance was markedly influenced by the purity of the enzymes used. Presence of compound III during turnover led to rapid inactivation of the peroxidases and, therefore, to both a lower yield of the sulphoxides and a lower enantioselectivity. Stopped‐flow kinetic data show that, for both LPO and CiP, the transition of compound I to compound II depends on the concentration of the methyl phenyl sulphide, suggesting an oxygen‐rebound mechanism. In line with this mechanism, a methyl phenyl sulphide radical cation was detected by EPR during turnover for LPO.
Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction
Authors: Pook Elisabeth , Grimm Silke , Bonin Angelika , Winkler Thomas , Hillen Wolfgang
Published: March 2003

We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against theTetR‐tet operator (tetO) complex. The epitopes of the anti‐TetR mAbs are localized in the α‐helix‐turn‐α‐helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface. The anti‐TetR‐Tc and one of the anti‐TetR‐tetO mAbs recognize epitopes near the Tc binding pocket. The other anti‐TetR‐tetO mAb binds to an epitope within the HTH. Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR‐Tc, or TetR‐tetO revealed different affinities of the mAbs for TetR in these functional states. Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR‐Tc indicating the same conformation in both forms. The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR‐Tc, supporting the idea of different conformations of that epitope in these forms of TetR. The greatest affinity differences were found for epitopes around the Tc binding pocket. Two anti‐TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR‐tetO and the lowest affinities for TetR‐Tc. The anti‐TetR‐Tc mAb has a discontinuous epitope, formed in TetR‐Tc, which is less well bound in TetR and not bound in the TetR‐tetO complex. One anti‐TetR‐tetO mAb does not recognize TetR‐Tc. Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.
A chicken homolog of mammalian interleukin‐1β : cDNA cloning and purification of active recombinant protein
Authors: Weining Kirsten C. , Sick Christine , Kaspers Bernd , Staeheli Peter
Published: March 2003

Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD‐11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC‐32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N‐terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin‐1β (ChIL‐1β). Northern blot analysis showed that ChIL‐1β RNA is quickly induced in blood monocyte‐derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL‐1β, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N‐terminus for easy purification by nickel chelate affinity chromatography. Purified His‐ChIL‐1β potently induced CXC chemokine RNA synthesis in CEC‐32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.
FhuF, an iron‐regulated protein of Escherichia coli with a new type of [2Fe‐2S] center
Authors: Müller Kerstin , Matzanke Berthold F. , Schünemann Volker , Trautwein Alfred X. , Hantke Klaus
Published: March 2003

We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K‐12. The FhuF protein was labeled with a His‐tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a [2Fe‐2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mössbauer parameters of FhuF obtained [oxidized protein as isolated : ΔEQ,4.2K = 0.474 mm s−1; Fe3+ (reduced protein) : ΔEQ = 0.978 mm s−1] are not typical of common [2Fe‐2S] proteins and indicate that FhuF has unusual structural properties.

The primary sequence of FhuF does not show any sequence similiarities to known [2Fe‐2S] proteins. By site‐directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe‐2S]Cys4 cluster. Mutants having the Cys‐to‐Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys‐Cys‐Xaa10‐Cys‐Xaa2‐Cys in FhuF differs considerably from the motif Cys‐Xaa2‐Cys‐Xaa9−15‐Cys‐Xaa2‐Cys found in other [2Fe‐2S] proteins. The unusual Cys‐Cys terminal group of the cluster may explain the atypical EPR and Mössbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.
Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities
Authors: Louërat‐Oriou Bénédicte , Perret Alain , Pompon Denis
Published: March 2003

Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH‐P‐450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P‐450‐reductases, ATR2 contrasted by its very low FMN affinity and required a thiol‐reducing agent for efficient cofactor binding to the FMN‐depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN‐binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P‐450 BM3 reductase. Affinities of reductases for rabbit P‐450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activies with artificial and natural (P‐450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P‐450 functions.
Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides
Authors: Meissner Harald , Liebl Wolfgang
Published: March 2003

A novel enzyme acting on starch and malto‐oligosaccharides was identified and characterised. The non‐hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto‐oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto‐oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X ± 2n (with X  3 and n = 0, 1, 2, ...). Maximum activity in a 10‐min assay was recorded at pH 6.5 and 85−90 +C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70 +C (pH 6.5, 0.34 mg ml−1 protein), MTase half‐lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded.

The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low‐level local similarity between MTase and the α‐amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (β/α)8‐fold as the core supersecondary structure which is typical for the α‐amylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the α‐amylase family, inserted between β‐strand 3 and α‐helix 3 of the (β/α)8‐barrel fold).
Cloning, properties and tissue distribution of natriuretic peptide receptor‐A of euryhaline eel, Anguilla japonica
Authors: Kashiwagi Masahide , Miyamoto Kentaro , Takei Yoshio , Hirose Shigehisa
Published: March 2003

During the course of cloning and characterization of natriuretic peptide receptor‐A (NPR‐A) from the euryhaline fish eel, Anguilla japonica, we identified a splice variant with unique structural properties that affect ligand‐inducible intrinsic guanylate cyclase activity. The variant, generated from a splice between a cryptic donor site and the normal acceptor site, lacked nine amino acid residues (VFTKTGYYK) in the kinase‐like regulatory domain. This deletion of a very short segment resulted in the complete loss of the ligand inducibility of the cyclase activity. The nine‐amino acid segment may therefore be useful as a target for studies aimed at clarifying the mechanism of activation of the guanylate cyclase domain. Characterization of the normal form of eel NPR‐A also led to the following interesting findings. Although eel NPR‐A had a domain structure very similar to that of mammalian counterparts, it lacked the third cysteine residue in the extracellular domain which is conserved among mammalian NPR‐A molecules. The eel receptor bound both amidated and nonamidated eel atrial natriuretic peptide (eANP) with high affinity but, when assayed for ligand‐inducible cGMP generation, it responded efficiently only to physiological concentrations of the amidated ligand, suggesting that the biologically active form is the amidated eANP, and the nonamidated form acts as a partial antagonist; similarly, nonhomologous rat ligands behaved like antagonists toward the eel receptor in the concentration range 0.1–10 nm. The receptor message was found to be relatively abundant in the osmoregulatory organs such as the gill, kidney, intestine and urinary bladder.
Structure and serological specificity of a new acidic O‐specific  polysaccharide of Proteus vulgaris O45
Authors: Bartodziejska Beata , Shashkov Alexander S. , Torzewska Agnieszka , Grachev Aleksei A. , Ziolkowski Andrzej , Paramonov Nikolay A. , Rozalski Antoni , Knirel Yuriy A.
Published: March 2003

The following structure of the O‐specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H‐ and 13C‐NMR spectroscopy, including two‐dimensional NOESY and H‐detected 1H, 13C heteronuclear multiple‐quantum coherence (HMQC) experiments:

Immunochemical studies, using rabbit polyclonal anti‐P. vulgaris O45 serum and LPS, OPS and Smith‐degraded OPS of P. vulgaris O45, showed the importance of β‐d‐GlcA in manifesting the serological specificity of the O‐antigen studied.
All three IκB isoforms and most Rel family members are stably associated with the IκB kinase 1/2 complex
Authors: Heilker Ralf , Freuler Felix , Pulfer Ruth , Di Padova Franco , Eder Jörg
Published: March 2003

Nuclear factor kappa B (NF‐κB) is an important transcription factor for the genes of many pro‐inflammatory proteins and is strongly activated by the cytokines interleukin‐1 and tumor necrosis factor (TNF)α under various pathological conditions. In nonstimulated cells, NF‐κB is present in the cytosol where it is complexed to its inhibitor IκB. Activation of NF‐κB depends on the signal‐induced phosphorylation of IκB by specific IκB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF‐stimulated and okadaic‐acid‐stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IκBα which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IκB kinases: the mitogen‐activated 90 kDa ribosomal S6 kinase (p90rsk1), the IκB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre‐assembled kinase–substrate complex in which the IKKs are directly or indirectly associated with several NF‐κB‐related and IκB‐related proteins: RelA, RelB, cRel, p100, p105, IκBα, IκBβ and IκBε. The existence of stable kinase–substrate complexes, the presence of all three known IκB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating IκBα‐, IκBβ‐ and IκBε‐derived peptides at the respective degradation‐relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF‐κB species.

 In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C‐terminal PEST sequence of IκBα, we observed efficient phosphorylation of serine 32 in IκBα by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.
Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH‐inducible fluorogenic substrate of trypanosomal cysteine proteinases
Authors: Serveau Carole , Lalmanach Gilles , Hirata Isaura , Scharfstein Julio , Juliano Maria A. , Gauthier Francis
Published: March 2003

The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl‐peptides based on the putative autoproteolytic sequence of the proteinase (VVG‐GP) located at the hinge region between the catalytic domain and the C‐terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz‐HPGGPQ‐EDDnp) which was also specifically cleaved by cruzipain (kcat/Km of 157 000 m–1·s–1) and by a homologous proteinase from Trypanosoma congolense. The pH activity profile of cruzipain on Abz‐HPGGPQ‐EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. The lack of activity at neutral and basic pH was due to a decrease in kcat, while the Km remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz‐HPGGPQ‐EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.
Decreases in cAMP phosphodiesterase activity in hepatocytes cultured with herbimycin A due to cellular microtubule polymerization related to inhibition of tyrosine phosphorylation of α‐tubulin
Authors: Ishibashi Kaname , Fujioka Toshiyuki , Ui Michio
Published: March 2003

The increase in cellular cAMP concentration during 10‐min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine‐phosphorylation of cellular proteins including α‐tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin‐induced tyrosine phosphorylation, and overcome by the addition to the 10‐min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4‐[3‐(cyclopentyloxy)‐4‐methoxyphenyl]‐2‐pyrrolidinone (rolipram) and 4‐(3‐butyloxy‐4‐methoxyphenyl)‐2‐imidazolidinone (Ro‐20‐1724, a PDE4 inhibitor), in addition to 3‐isobutyl‐1‐methylxanthine (a nonselective inhibitor). Rapid breakdown of the once‐accumulated cAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 µm during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule‐disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine‐containing medium was further supplemented with taxol, a microtubule‐stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol‐like and vinblastine‐suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein‐bound anti‐(α‐tubulin). Thus, 4‐h culture of hepatocytes with herbimycin inhibits phosphorylation of the C‐terminal tyrosine residue of α‐tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism.
Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis
Authors: Mak Chi‐ho , Ko Ronald C.
Published: March 2003

Double‐stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double‐stranded breaks to the native DNA. The ES double‐stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective‐stage larvae of T. spiralis collected at 37 °C and was present in much smaller amounts in samples collected at 43 °C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated ≈ 25, 30 and 58 kDa, respectively.
Nucleoside diphosphate kinase activity in soluble transducin preparations
Authors: Klinker Jan F. , Seifert Roland
Published: March 2003

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17–23‐kDa subunits and catalyze the reaction N1TP + N2DP → N1DP + N2TP via formation of a histidine‐phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP‐binding proteins (G‐proteins) by catalyzing the formation of GTP from GDP, but the properties of G‐protein‐associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G‐protein transducin. The NDPK is operationally referred to as transducin‐NDPK. Like known NDPKs, transducin‐NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin‐NDPK than ADP and CDP, and guanosine 5′‐[γ‐thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5′‐[γ‐thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin‐NDPK. Guanosine 5′‐[β,γ‐imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [γ‐32P]ATP and [γ‐32P]GTP [32P]phosphorylated, and [35S]ATP[S][35S]thiophosphorylated, a 36‐kDa protein comigrating with transducin‐β. p[NH]ppG potentiated [35S]thiophosphorylation of the 36‐kDa protein. 32P‐labeling of the 36‐kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17–23‐kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP‐prefering and guanine nucleotide‐regulated NDPK; (b) transducin‐β may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin‐NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.
Expression of receptors for human angiogenin in vascular smooth muscle cells
Authors: Hatzi Elissavet , Badet Josette
Published: March 2003

Human angiogenin is a plasma protein with angiogenic and ribonucleolytic activities. Angiogenin inhibited both DNA replication and proliferation of aortic smooth muscle cells. Binding of 125I‐angiogenin to bovine aortic smooth muscle cells at 4 °C was specific, saturable, reversible and involved two families of interactions. High‐affinity binding sites with an apparent dissociation constant of 0.2 nm bound 1 × 104 molecules per cell grown at a density of 3 × 104·cm−2. Low‐affinity binding sites with an apparent dissociation constant of 0.1 μm bound 4 × 106 molecules·cell−1. High‐affinity binding sites decreased as cell density increased and were not detected at confluence. 125I‐angiogenin bound specifically to cells routinely grown in serum‐free conditions, indicating that the angiogenin‐binding components were cell‐derived. Affinity labelling of sparse bovine smooth muscle cells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250–260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Together these data suggest that cultured bovine and human aortic smooth muscle cells express specific receptors for human angiogenin.
Thermal effects on an enzymatically latent conformation of coagulation factor VIIa
Authors: Petersen Lars C. , Persson Egon , Freskgård Per‐Ola
Published: March 2003

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 °C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891–18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N‐terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N‐terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N‐terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross‐linked factor VIIa–tissue factor complex; this showed that cross‐linking stabilized the active conformation of factor VIIa and essentially prevented its thermally‐induced transformation into the inactive state.
Aspects of the molecular structure and dynamics of neuropeptide Y
Authors: Nordmann Antje , Blommers Marcel J. J. , Fretz Heinz , Arvinte Tudor , Drake Alex F.
Published: March 2003

Human neuropeptide Y (hNPY) and the Q34→P34 mutant (P34‐hNPY) have been characterized by CD spectroscopy. hNPY self‐associates in aqueous solution with a dimerization constant in the micromolar range. The self‐association correlates with an increase in secondary‐structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5–84 °C) and in ethanediol/water (2 : 1) (−90 ° to +90 °C). A single‐residue mutation, Q34→P34, affects the pH, thermal and self‐association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP‐fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.
High‐resolution A‐DNA crystal structures of d(AGGGGCCCCT)
Authors: Gao Yi‐Gui , Robinson Howard , Wang Andrew H.‐J.
Published: March 2003

A‐DNA conformation is favored by guanine‐rich sequences, such as (dG)n·(dC)n, or under low‐humidity conditions. Earlier A‐DNA crystal structures revealed some conformational variations which may be the result of sequence‐dependent effects and/or crystal packing forces. Here we report the high‐resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n·(dC)n sequence. The P212121 form has been analyzed using data to 1.1 Å resolution by the anisotropic temperature factor refinement procedure of the shelx97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A‐DNA structure. The backbone torsional angles fall in a narrow range, except for the α/γ angles which have two distinct combinations (gauche−/gauche+ or trans/trans). An A‐DNA model of poly(dG)·poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A‐DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A‐DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.
Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of protein phosphatase‐1
Authors: Ceulemans Hugo , Van Eynde Aleyde , Pérez‐Callejón Encarna , Beullens Monique , Stalmans Willy , Bollen Mathieu
Published: March 2003

sds22 is a regulatory subunit of protein phosphatase‐1 that is required for the completion of mitosis in yeast. It consists largely of 11 tandem leucine‐rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including protein phosphatase‐1. In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7. This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine‐rich repeats. Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT‐PCR and Northern blot analysis. The sds22 transcripts are expected to encode four different polypeptides. sds22α1 corresponds to the variant cloned previously from human brain [Renouf et al. (1995) FEBS Lett. 375, 75–78]. Sds22β1 is truncated within the ninth repeat and has a short and different C‐terminus. Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22α2 and sds22β2. The 5′‐flanking region of PPP1R7 contains two NF‐Y‐binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c‐Myb, Ik‐2 and NF‐1, which are conserved in the mouse gene.
Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity
Authors: Eberhardt Ines , Cederberg Håkan , Li Haijuan , König Stephan , Jordan Frank , Hohmann Stefan
Published: March 2003

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Δ strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1‐8 and pdc1‐14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild‐type by a single amino acid. The cloned pdc1‐8 and pdc1‐14 alleles, as well as the in‐vitro‐generated pdc1‐51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1‐8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1‐8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Δ mutation. The third mutation, pdc1‐803, led to a deletion of two amino acids adjacent to the pdc1‐8 mutation. The alleles pdc1‐8 and pdc1‐803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1‐8 (Asp291Asn) and the two amino acids deleted in pdc1‐803 (Ser296 and Phe297) are located within a flexible loop in the β domain. This domain appears to determine the relative orientation of the α and γ domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1‐14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1‐8 and pdc1‐14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.
Binding characteristics of an anti‐Siaα2–6GalNAcα‐Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132)
Authors: Tanaka Nobuhiro , Nakada Hiroshi , Inoue Mizue , Yamashina Ikuo
Published: March 2003

To determine the epitopic structure for an anti‐Siaα2–6GalNAcα‐Ser/Thr (anti‐sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column‐bound fraction. On solid‐phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe‐Ser*‐Gly‐Glu‐Thr*‐Ser*‐Thr*‐Thr*‐Val‐Ile‐Ser*‐Gly‐Thr*‐Asn‐Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.
C‐CAM‐mediated adhesion leads to an outside‐in dephosphorylation signal
Authors: Lucka Lothar , Budt Matthias , Cichocka Iwona , Danker Kerstin , Horstkorte Rüdiger , Reutter Werner
Published: March 2003

The rat cell–cell adhesion molecule C‐CAM, a member of the carcinoembryonic antigen family, was shown to be expressed in various isoforms, differing in the length of the cytoplasmic domain. The long isoform C‐CAML inhibits the growth of different malignant cells. Several studies suggest that it is involved in the mechanism of signal transduction. So far no direct correlation between C‐CAM function and C‐CAM phosphorylation has been reported. In the present study we addressed the question of whether C‐CAM‐mediated adhesion is accompanied by changes in phosphorylation of the cytoplasmic domain of C‐CAM. It was demonstrated that C‐CAML is constitutively phosphorylated in adherent growing cells as well as in cells growing in suspension. In contrast, C‐CAML‐mediated cell aggregation is accompanied by a 40% reduction in C‐CAML phosphorylation compared with nonaggregated cells. The same dephosphorylation was achieved by antibody‐induced clustering of C‐CAML in the plasma membrane. Phosphorylation and dephosphorylation indicate a C‐CAM‐mediated outside‐in signalling induced by cell–cell adhesion.
Preferential uptake and accumulation of oxidized vitamin C by THP‐1 monocytic cells
Authors: Laggner Hildegard , Besau Veronika , Goldenberg Hans
Published: March 2003

THP‐1 cells preferentially accumulate vitamin C in its oxidized form. The uptake displays first‐order kinetics and leads to a build‐up of an outward concentration gradient which is stable in the absence of extracellular vitamin.

 The transport is faster than reduction by extracellular glutathione or by added cytosolic extract, and glutathione‐depleted cells show the same uptake rates as control cells. In addition, energy depletion or oxidation of intracellular sulfhydryls does not inhibit accumulation of ascorbate. The accumulation, however, always occurs in the reduced form.

 The affinity for dehydroascorbate is lower (Km 450 µm vs 60 µm) than for reduced ascorbate, but the maximal rate is more than 30 times higher (581 compared to 19 pmol·min−1 per 106 cells), and it is independent of sodium, whereas the uptake of ascorbate is not. The sodium gradient also allows accumulation of reduced ascorbate.

 Inhibitors of glucose transport by the GLUT‐1 transporter also inhibit uptake of dehydroascorbate (DHA), but there are some inconsistencies, because the Ki‐values are higher than reported for the isolated transporter and one inhibitor (deoxyglucose) is noncompetitive.

 The preferential uptake of the dehydro‐form of the vitamin may be useful for situations where this short‐lived metabolite is formed by oxidation in the environment.
The effect of intracellular iron concentration and nitrogen monoxide on Nramp2 expression and non‐transferrin‐bound iron uptake
Authors: Wardrop S. L. , Richardson D. R.
Published: March 2003

Recent studies have demonstrated that the protein product (natural resistance associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts as an Fe transporter involved in the uptake of Fe from transferrin (Tf) and low Mr Fe complexes. Interestingly, there are two splice variants of Nramp2, one with a putative iron‐responsive element (IRE) in its 3′ untranslated region (UTR) and another without. Due to the importance of Nramp2 in Fe transport, and the presence of an IRE in its 3′‐UTR, we have examined the effect of Fe‐deprivation, Fe‐loading, and nitrogen monoxide on the expression of Nramp2 mRNA. These results were compared to the expression of transferrin receptor (TfR) mRNA which also has IREs in its 3′‐UTR and is regulated by Fe and NO via the binding of iron‐regulatory proteins (IRPs) to its IREs. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in lysates from a mouse fibroblast cell line (LMTK−). Moreover, reverse transcription‐PCR (RT‐PCR) demonstrated that both the IRE and non‐IRE‐containing transcripts were present within these cells. However, there was no change in Nramp2 mRNA expression in LMTK− cells after a 20‐h incubation with either the Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrate (FAC), or the NO generator, S‐nitroso‐N‐acetylpenicillamine (SNAP). In contrast, these agents caused a marked change in the RNA‐binding activity of the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caused an appropriate change in [59Fe] uptake from [59Fe]Tf, viz., an increase in Fe uptake after exposure to DFO and a decrease after treatment with FAC. As Nramp2 can transport Fe from non‐Tf‐bound Fe, the effect of preincubation with DFO and FAC was also examined on Fe uptake from [59Fe]nitrilotriacetate and [59Fe]citrate. However, in contrast to the results found for [59Fe]Tf, incubation with DFO and FAC did not result in appropriate regulation of Fe uptake from [59Fe]nitrilotriacetate or [59Fe]citrate. These data demonstrate that non‐Tf‐bound Fe uptake was not under control of the IRP‐IRE system in these cells. Collectively, the results indicate that in LMTK‐fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA.