bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stoichiometry, Organisation and Catalytic Function of Protein X of the Pyruvate Dehydrogenase Complex from Bovine Heart
Authors: Sanderson Sanya J. , Miller Clare , Lindsay J. Gordon 
Published: August 2004

Mammalian pyruvate dehydrogenase complex (PDC) contains a subunit, protein X, which mediates high‐affinity binding of dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide acetyltransferase (E2) core. Precise stoichiometric determinations on bovine heart PDC, by means of two approaches, indicate the presence of 12 mol protein X/mol PDC and 60 mol E2/mol PDC.Studies of the organisation of collagenase‐modified PDC by means of covalent cros‐linking of N,N′‐1,2‐phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross‐linked products have Mr values corresponding to homodimers of protein X. However, significant formation of higher‐Mr aggregates indicates that lipoyl domains of protein X can form an interacting network independent of E2 lipoyl domains. These data suggest that either 12 interacting X monomers or 6 interacting X dimers are involved in the binding of six E3 homodimers to the E2/X core. The presence of 60 E2 subunits/complex also supports proposals for a non‐integrated external position of protein X.Collagenase‐treated PDC possesses residual activity (15%), indicating that protein‐X‐linked lipoamide groups can substitute for the lipoyl domains of E2 in overall complex catalysis. Protein‐X‐mediated diacetylation of dihydrolipoamide moieties is also performed by the modified complex which raises the possibility of a unique catalytic function for protein X.
Molecular Characterisation of Plant Endoplasmic Reticulum
Authors: Coughlan Sean J. , Hastings Craig , Winfrey Ron J. 
Published: August 2004

Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two‐step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components.A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity‐purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide‐isomerase. Northern and Southern blot analysis showed that the mRNA from a single‐copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide‐isomerase catalytic activity, thus confirming identity of this protein.
Catabolic Ornithine Carbamoyltransferase of Pseudomonas aeruginosa
Authors: Nguyen Van Thanh , Baker Darren P. , Tricot Catherine , Baur Heinz , Villeret Vincent , Dideberg Otto , Gigot Daniel , Stalon Victor , Haas Dieter 
Published: August 2004

Pseudomonas aeruginosa, has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu105 and the C‐terminus are known to be essential for this cooperativity. When Glu105 and nine C‐terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis‐Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF, gene to the P. aeruginosa arcB, gene. A hybrid enzyme consisting of 101 N‐terminal ArgF amino acids fused to 233 C‐terminal ArcB residues and the reciprocal ArcB‐ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N‐terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N‐terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N‐terminal ArgF amino acids and the corresponding C‐terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N‐terminal ArcB region, a replacement of motif 28–33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three‐dimensional structure of catabolic OTCase, which allows predictions about trimer–trimer interactions. Dodecameric assembly appears to require at least three regions: the N‐ and C‐termini (which are close to each other in a monomer), residues 28–33 and residues 147–154.Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu105→Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.
Quantitative Analysis of the Targeting of Mannose‐Terminal Glucocerebrosidase
Authors: Bijsterbosch Martin K. , Donker Wilma , Van De Bilt Hendrika , Van Weely Sonja , Van Berkel Theo J. C. , Aerts Johannes M. F. G. 
Published: August 2004

Gaucher's disease is an inherited lysosomal storage disorder that is caused by a deficiency of glucocerebrosidase. The resulting accumulation of the substrate glucosylceramide in macrophages of liver, spleen, and bone marrow causes severe clinical symptoms. Gaucher's disease is treated by intravenous administration of a modified glucocerebrosidase (Alglucerase), which has exposed mannose residues to promote uptake by target macrophages. To evaluate the effectiveness of the targeting of Alglucerase, we studied the fate of the enzyme in the rat. Intravenously injected Alglucerase was rapidly cleared from the circulation (half‐life 2.0±0.5 min). The liver was the main site of uptake, with 65.6±1.2% of the dose present at 10 min after injection. Smaller amounts (<3% of the dose) were taken up by spleen and bone marrow. Previous injection with mannan substantially increased the plasma half‐life of the enzyme (14.8±3.2 min versus 1.7±0.3 min in solvent‐preinjected controls) and uptake of the enzyme by liver, spleen and bone marrow was reduced by >90%. These findings indicate that the enzyme is taken up by these organs via mannose‐specific receptors. Subcellular fractionation of the liver indicated that the enzyme is internalized and transported to the lysosomes. By isolating various liver cell types after injection of the Alglucerase, it was found that endothelial cells are the main site of uptake of the enzyme: 60.8±3.4% of the total liver uptake. Parenchymal and Kupffer cells were responsible for 31.0±3.1% and 8.2±0.7% of the hepatic uptake, respectively.We conclude that Alglucerase is rapidly cleared from the circulation by mannose‐specific receptors in liver, spleen, and bone marrow. However, less than 10% of the enzyme taken up by the liver is accounted for by Kupffer cells, the hepatic target cells for therapeutic intervention. It is suggested that alterations of the formulation of the therapeutic enzyme may lead to a higher uptake by Kupffer cells and other macrophages, and thus to a more (cost)effective therapy of Gaucher's disease.
Energizing Effects of Illumination on the Reactivities of Lysine Residues of the γ Subunit of Chloroplast ATP Synthase
Authors: Komatsu‐Takaki Mizuho 
Published: August 2004

Incubation of chloroplast thylakoids with pyridoxal 5′‐phosphate for a short time (5 s) modified the lysine residues of the γ subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s after the onset of illumination. Illumination of thylakoids increased the reactivities of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20–30 s after the onset of illumination. In the presence of 10 mM NH4CI, illumination of thylakoids did not change the reactivities of these lysine residues.These results suggest that the Lys24‐ and Lys30‐containing region of the γ subunit changes its conformation rapidly in response to μH+ and that the Lys222‐ and Lys231‐containing region of the γ subunit changes its conformation in two steps in response to μH+ formation.
Role of Insulin‐Like Growth Factors in Autocrine Growth of Human Retinoblastoma Y79 Cells
Authors: Giuliano Michela , Vento Renza , Lauricella Marianna , Calvaruso Giuseppe , Carabillo' Maria , Tesoriere Giovanni 
Published: August 2004

In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin‐like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross‐linking procedure, that Y79 cells contain the type‐I IGF receptor (IGF‐IR). Furthermore, surface‐bound IGF‐I is internalised by the receptor, then degraded to amino acids.Insulin, IGF‐I and IGF‐II caused down‐regulation of IGF‐IR; the effect is concentration and time dependant. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF‐I while the apparent Kd value calculated for IGF‐I binding was not significantly modified. IGF‐I, IGF‐II and insulin induced tyrosine phosphorylation of IGF‐IR. Tyrosine phosphorylation of this receptor with, however, a less strong signal, was detectable even in cells cultured in serum‐free medium without the addition of any exogenous growth factor. Similar results have been found concerning the tyrosine phosphorylation of insulin receptor substrate‐1 (IRS 1). Tyrosine phosphorylation of both IGF‐IR and IRS 1, either under basal conditions or after stimulation with growth factors, was strongly inhibited when α‐IR3, a monoclonal antibody to IGF‐IR, was added to the culture.IGF‐I was capable of inducing Y79 cell proliferation and its effect was entirely inhibited by the addition of α‐IR3. This antibody also markedly reduced the proliferation of Y79 cells cultured in serum‐free medium not supplemented with stimulatory factors. Our results indicate that IGF‐I and IGF‐IR mediate an autocrine growth mechanism in Y79 cells.
A Comparative study of the Phosphotyrosyl Phosphatase Specificity of Protein Phosphatase Type 2A and Phosphotyrosyl Phosphatase Type 1B Using Phosphopeptides and the Phosphoproteins p50/HS1, c‐Fgr and Lyn
Authors: Agostinis Patrizia , Donella‐Deana Arianna , Van Hoof Christine , Cesaro Luca , Brunati Anna Maria , Ruzzene Maria , Merlevede Wilfried , Pinna Lorenzo A. , Goris Jozef 
Published: August 2004

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl‐phosphatase‐activator‐(PTPA)‐stimulated protein phosphatase (PP)2AD (rabbit muscle) and a bona fide PTP‐1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine‐containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c‐Src and c‐Fgr, as well as the intact phosphoprotein p501 HS1 and the Src‐related tyrosine kinases, Lyn and c‐Fgr.The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2AD than for PTP‐1B, C‐terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2AD, but they do not affect the PTP‐1B activity. Acidic residues at the –2 and –3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP‐1B.The higher‐order structure of the protein substrates also differentially influences both enzymes: the phospho‐octapeptide KDDEYp NPA, which reproduces the autophosphorylation site in c‐Fgr (Tyr400), is only dephosphorylated by PP2AD if embedded in the intact protein, whereas the opposite is true for PTP‐1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP‐1B and not for PP2AD. Lyn and c‐Fgr phosphorylated by C‐terminal Src kinase (CSK) at their down‐regulatory site are resistant to the action of both PTPases while the [Phe6]Src‐(514–533) phosphopeptide, representing the highly similar site affected by CSK in c‐Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c‐Fgr Tyr400 site by PP2AD is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down‐regulatory site) of c‐Fgr are phosphorylated, PP2AD can reverse both phosphorylations.
X‐ray Absorption Spectroscopy Study of Zinc Coordination in Tetanus Neurotoxin, Astacin, Alkaline Protease and Thermolysin
Authors: Morante Silvia , Furenlid Lars , Schiavo Giampietro , Tonello Fiorella , Zwilling Robert , Montecucco Cesare 
Published: August 2004

Tetanus and botulinum neurotoxins constitute a new group of Zn‐endopeptidases which has recently been actively investigated with the purpose of correlating their biochemical properties to their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn‐endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and a glutamate‐bound water molecule. The two histidines and the glutamate residues belong to the HEXXH motif which is characteristic of most Zn‐endopeptidases. A fourth metal ligand is a glutamate in thermolysin‐like proteinases, but it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X‐ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin.In this work X‐ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near‐edge and extended fine‐structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one water molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.
Reconstitution and Pigment‐Binding Properties of Recombinant CP29
Authors: Giuffra Elisabetta , Cugini Daniela , Croce Roberta , Bassi Roberto 
Published: August 2004

The minor light‐harvesting chlorophyll‐a/b ‐binding protein CP29 (Lhcb4), overexpressed in Esche‐richia coli, has been reconstituted in vitro with pigments. The recombinant pigment‐protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light‐harvesting chlorophyll‐alb‐binding protein of photosystem II (LHCII).The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll‐a/b ‐content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146–150; Paulsen, H., Riimler, U. & Riidiger, W. (1990) Planta (Heidelb.) 181, 204–211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.
Occurrence and Structural Analysis of Highly Sulfated Multiantennary N‐linked Glycan Chains Derived from a Fertilization‐Associated Carbohydrate‐Rich Glycoprotein in Unfertilized Eggs of Tribolodon hakonensis
Authors: Taguchi Tomohiko , Iwasaki Mariko , Muto Yutaka , Kitajima Ken , Inoue Sadako , Khoo Kay‐Hooi , Morris Howard R. , Dell Anne , Inoue Yasuo 
Published: August 2004

This study represents the first detailed investigation of the nature of highly sulfated (keratan‐sulfate‐like) complex‐type asparagine‐linked glycans having a tetraantennary core structure and shows the effectiveness of fast‐atom‐bombardment mass spectrometric (FAB‐MS) methods incorporating derivatization and mild methanolysis for analyzing such complex types of sulfated glycans.The structure of the N‐glycan chains was unambiguously established by a combination of compositional analysis, methylation analysis, mild methanolysis for desulfation, hydrazinolysis/nitrous acid deamination, enzymatic (endo‐β‐galactosidase and peptide: N‐glycosidase F) digestions, and instrumental analyses (1H‐NMR spectroscopy and FAB‐MS) which revealed the novel repeating sulfated carbohydrate sequences, ±Galβ1 → 4Galβ1[→ 4(HSO3→ 6)GlcNAcβ1 → 3(±Galβ1 → 4)Galβ1]n→ (see Structure I; p+q+r+s ≈ 14). This sequence is unique in: (a) the skeletal structure is similar to that of keratan sulfate but is completely devoid of 6‐O‐sulfated Gal residues and (b) the presence of branched Gal residues in the sequence → 4GlcNAcβ1 → 3(Galβ1 → 4)Galβ1→.
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11β‐Hydroxylase Activity in Recombinant Yeast Mitochondria
Authors: Dumas Bruno , Cauet Gilles , Lacour Thierry , Degryse Eric , Laruelle Laurence , Ledoux Catherine , Spagnoli Roberto , Achstetter Tilman 
Published: August 2004

In mammals, the final 11β‐hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P‐45011β, together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11β‐hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P‐45011β whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH‐adrenodoxin‐reductase‐like activity. Adrenodoxin and P‐45011β behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P‐45011β. The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH.The notion that a yeast microsomal NADPH P‐450 oxidoreductase can transfer electrons to mammalian microsomal P‐450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P‐450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.
Study of the Coordination Chemistry of Prostaglandin G/H Synthase by Resonance Raman Spectroscopy
Authors: Gaspard Sarra , Chottard Geneviève , Mahy Jean‐Pierre , Mansuy Daniel 
Published: August 2004

Resonance Raman spectra of prostaglandin G/H synthase (PGHS) in its ferric and ferrous states have been obtained by Soret excitation. In native PGHS, which contained only 0.25 heme/monomeric apoprotein, the ferric heme was in a high‐spin hexacoordinated state. The presence of a vibration at 489 cm‐1 that was responsive to H216O→H218O replacement was taken as evidence for the presence of a H‐bonded H2O molecule as the sixth ligand of the Fe. A study, by CD and resonance Raman spectroscopy, of heme incorporation into the apoprotein showed that, for heme/protein ratios lower than 0.5, the heme was in the same ferric high‐spin hexacoordinated state as in the native enzyme. For heme/protein ratios higher than 0.5, the concomitant formation of two minor species was observed: a low‐spin hexacoordinated species which could be due to the axial coordination of a distal histidine to the Fe trans to its proximal histidine ligand; and a high‐spin pentacoordinated species that corresponded to non‐specific binding of the heme to the apoprotein.In the reduced state, the heme of PGHS contained a high‐spin pentacoordinated Fe(II) with a histidine as the proximal ligand. However, this species shifted spontaneously towards a low‐spin hexacoordinated Fe(II) species in which the iron was probably coordinated by a distal histidine as the sixth axial ligand. The PGHS Fe(II) · CO derivative displayed an Fe‐CO stretching mode at 529 cm−1, which is in the range observed for peroxidases. Such a high frequency could be due to H‐bonding between the oxygen atom of the CO ligand and the distal histidine, His207. Since this histidine plays an important role, by coordination of Fe(II) or Fe(III) of PGHS and stabilization of the ligands of the Fe, H2O or CO by H‐bonding, it is suggested that this histidine could also play a key role in the cleavage of the O‐O bond of peroxides by peroxidases.
Structural Studies of the O‐Specific Polysaccharide of Hafnia alvei Strain 1209 Lipopolysaccharide
Authors: Niedziela Tomasz , Petersson Carl , Helander Anne , Jachymek Wojciech , Kenne Lennart , Lugowski Czeslaw 
Published: August 2004

The structure of the O‐specific side chains of the Hafnia alvei strain 1209 lipopolysaccharide has been investigated. Methylation analysis and 1H‐NMR and 13C‐NMR spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of pentasaccharide repeating units that have the following structure:
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The relative intensity of the signals from the terminal repeating unit in the 1H‐NMR spectrum, the amount of 2,3,6‐tri‐O ‐methylgalactose in the methylation analysis, and the matrix‐assisted laser‐desorption ionisation time‐of‐flight (MALDI‐TOF) mass spectrum of the O‐polysaccharide indicated that the structure is also the biological repeating unit and that the O‐chains mainly consisted of 8–11 repeating units and, on average, ten repeating units.