bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression in Escherichia coli of a secreted invertebrate ferritin
Authors: VON DARL Matthias , HARRISON Pauline M. , BOTTKE Werner 
Published: March 2005

The coding regions of the cDNAs for cytoplasmic soma ferritin and secreted yolk ferritin from the snail Lymnaea stagnalis were inserted into the prokaryotic expression vector pEMBLex2. The vector directed the synthesis in Escherichia coli of soma ferritin up to a concentration of 15% of soluble proteins. Soma ferritin was expressed as the multimeric protein (480 kDa). Its similarity with natural soma ferritin was confirmed by PAGE, immunostaining and electron microscopy. Yolk ferritin was expressed in the form of inclusion bodies. Attempts to refold and assemble the purified yolk ferritin subunit in vitro failed. The yolk ferritin coding sequence was therefore inserted into the expression vector pMAL‐p2.At a growth temperature of the bacterial cells of 23°C and at an isopropyl β‐d‐thiogalactopyranoside concentration of 50 μM, about 5% of the induced MalE‐yolk‐ferritin fusion protein was secreted into the periplasmic space and could be purified by affinity chromatography on amylose; the rest occurred as insoluble cytoplasmic inclusion bodies. Soluble MalE–yolk‐ferritin fusion protein was capable of assembly into ferritin‐like particles. Fully assembled yolk apoferritin shells (610 kDa) were obtained by digestion of these particles with proteinase K (yield: 180 μg yolk ferritin/l bacterial culture). Recombinant yolk ferritin was capable of taking up iron in vitro. Yolk ferritin (610 kDa) and soma ferritin (480 kDa) were run to the pore limit of a non‐denaturing 5–20% PAGE gradient gel. Under these conditions, yolk ferritin had a higher mobility than soma ferritin (480 kDa) and therefore the yolk ferritin may have a rather compact structure. A 41‐amino‐acid‐residue stretch of the insertion, a distinctive feature of the yolk ferritin subunit, was deleted by site‐directed mutagenesis. The MalE–yolk‐ferritin variant thus obtained was readily degradable by proteinase K and could not be assembled into ferritin‐like particles. Therefore residues in the deleted peptide must be important for the maintenance of the native structure.
Glucokinase expression in rat hepatoma cells induces glucose uptake and is rate limiting in glucose utilization
Authors: VALERA Alfons , BOSCH Fatima 
Published: March 2005

In contrast to hepatocytes, hepatoma cells lack glucokinase activity and show increased aerobic glycolysis. FTO‐2B and H4IIE rat hepatoma cell lines were obtained in which the rat glucokinase gene was expressed (FTOGK and H4GK). These lines were generated by infection of the hepatoma cells with a retroviral vector carrying the phosphoenolpyruvate carboxykinase (PEPCK)‐glucokinase chimeric gene. Both the FTOGK and H4GK cells expressed the chimeric gene in a regulated manner, like the endogenous PEPCK gene. Glucokinase activity was detected in both FTOGK and H4GK. These cell lines showed a marked increase in glucose uptake with 18.5 mM glucose in the incubation medium. FTOGK and H4GK showed an increase in the content of glucose 6‐phosphate, and were able to accumulate high levels of glycogen, in contrast to FTO‐2B cells, which were unable to store the polysaccharide. In addition, cells expressing glucokinase showed high concentration of fructose 2,6‐bisphosphate and substantial lactate production, which was related to the glucose concentration in the medium and the time of incubation.These results suggest that glucose phosphorylation is rate limiting for glucose uptake and utilization in FTO‐2B and H4IIE cells.
Optical spectrum of myeloperoxidase
Authors: FLORIS René , KIM Younkyoo , BABCOCK Gerald T. , WEVER Ron 
Published: March 2005

The optical spectrum of reduced myeloperoxidase (EC 1.11.1.7) displays an unusual red shift of the Soret band which is at 472 nm and the α‐band which is at 636 nm. The spectral properties of myeloperoxidase can be modified by means of acid treatment. Upon short exposure to acid (pH 1.7) the red‐shifted optical absorption spectrum of the reduced enzyme (λmax at 472 nm) was blue‐shifted (λmax at 448 nm) but the spectrum of the reduced state could be restored by increasing the pH. By contrast, the resonance Raman spectra of both the oxidized and reduced enzyme are essentially the same at both pH 1.7 and pH 7.0. This shows that the optical spectrum and the resonance Raman spectrum are not directly correlated, which we interpret to indicate that the reversible effects of lower pH primarily affect the excited‐state energy levels of the macrocycle. The EPR spectrum of the oxidized enzyme showed a reversible conversion from a high‐spin rhombic spectrum (gx= 6.7, gy= 5.2) at neutral pH into a more axial high‐spin spectrum (gx=gy= 5.8) at low pH.Upon prolonged exposure to acid (20 min) optical absorbance spectra, EPR spectra, resonance Raman spectra and the chlorinating activity were irreversibly affected. We propose that a negatively charged protonatable residue in the proximity of a pyrrole nucleus of the haem group is present that imposes the red shift in the optical absorption spectrum. This is consistent with the available X‐ray structure data.
Post‐translational modifications in aspartate aminotransferase from Sulfolobus solfataricus
Authors: ZAPPACOSTA Francesca , SANNIA Giovanni , SAVOY Luc‐Alain , MARINO Gennaro , PUCCI Piero 
Published: March 2005

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments.The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast‐atom‐bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N‐ɛ‐methyl derivatives to a substoichiometric extent.
Characterization of acyl‐CoA thioesterase activity in isolated rat liver peroxisomes
Authors: WILCKE Mona , ALEXSON Stefan E. H. 
Published: March 2005

A common function of peroxisomes in eukaryotic cells is β‐oxidation of fatty acids. In animal cells, β‐oxidation is compartmentalized to peroxisomes and mitochondria. Although regulation of β‐oxidation in mitochondria has been extensively studied, knowledge on its regulation in peroxisomes is still limited. We have considered the possibility that peroxisomes may contain acyl‐CoA thioesterases with different substrate specificities that possibly regulate metabolism of different lipids by regulation of substrate availability. In the present study, we have investigated the presence of short‐chain and long‐chain acyl‐CoA thioesterase activities in rat liver peroxisomes.Light‐mitochondrial fractions, enriched in peroxisomes, were fractionated by Nycodenz density gradient centrifugation and gradient fractions were analyzed for acyl‐CoA thioesterase and marker enzyme distributions. Fractionation of livers from normal rats showed that most of the long‐chain acyl‐CoA thioesterase activity was localized in microsomes and mitochondria, and only low activity was found in fractions containing peroxisomes. The gradient distribution of propionyl‐CoA thioesterase activity showed this activity to be localized mainly in mitochondria and in fractions possibly representing lysosomes, with a small peak of activity in peroxisomal fractions.Di(2‐ethylhexyl)phthalate treatment induced the specific propionyl‐CoA thioesterase activity approximately threefold in the peak mitochondrial fractions and about onefold in peroxisomal fractions; the activity appeared to be almost exclusively localized to these organelles. The specific activity of myristoyl‐CoA thioesterase was induced 1–2‐fold in peroxisomal peak fractions and more than 10‐fold in the mitochondrial peak fraction, whereas it was unchanged in microsomes.The chain‐length specificity of acyl‐CoA thioesterase activity in isolated peroxisomes suggests that peroxisomes contain an inducible short‐chain thioesterase active on C2‐C4 acyl‐CoA species (possibly a ‘propionyl‐CoA’ thioesterase). In addition, peroxisomes contain medium‐chain to long‐chain thioesterase activity, probably due to separate enzymes based on the different chain‐length specificities observed in peroxisomes from normal and di(2‐ethylhexyl)phthalate‐treated rats.A long‐chain acyl‐CoA thioesterase was partially purified from isolated peroxisomes and found to be active only on fatty‐acyl‐CoA species longer than octanoyl‐CoA. The protein is apparently a monomer of about 40 kDa and clearly different from microsomal long‐chain acyl‐CoA thioesterase. An induction of this long‐chain thioesterase may explain the observed change in chain‐length specificity in peroxisomes isolated from normal and di(2‐ethylhexyl)phthalate‐treated rats.Possible physiological functions of these thioesterases are discussed.
Purification of a bone sialoprotein‐binding protein from Staphylococcus aureus
Authors: YACOUB Alia , LINDAHL Per , RUBIN Kristofer , WENDEL Mikael , HEINEGÅRD Dick , RYDÉN Cecilia 
Published: March 2005

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C., Maxe, I., Franzén, A., Ljungh, Å., Heinegård, D. & Rubin, K. (1987) Lancet II, 515]. We now report on the purification of a cell‐wall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell‐wall components with capacity to inhibit binding of 125I‐labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0).Preparative SDS/PAGE and protein‐overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with Mr 75000–110000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono‐Q anion‐exchange column. Inhibitory activity was eluted at 0.6–0.8 M NaCl and could be further purified by affinity chromatography on BSP‐Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity.Analysis by SDS/PAGE revealed a single Mr 97000 polypeptide in the eluate. The purified Mr 97000 protein bound BSP in protein‐overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the Mr 97000 protein. Our data demonstrate the presence of a S. aureus, cell‐surface BSP‐binding protein. This protein could be involved in bacterial tropism in osteomyelitis.
Mast cell proteinases activate precursor forms of collagenase and stromelysin, but not of gelatinases A and B
Authors: LEES Martyn , TAYLOR David J. , WOOLLEY David E. 
Published: March 2005

Mast cell activation in vivo is often associated with areas of oedema and connective‐tissue degradation. Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components of the extracellular matrix. The matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP‐1), stromelysin (MMP‐3), gelatinase A (MMP‐2) and gelatinase B (MMP‐9).Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP‐1 and MMP‐3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP‐1 and pMMP‐3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP‐3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP‐2 and MMP‐9. However, MMP‐3 previously activated by mast cell proteinases was shown to activate pMMP‐9, but not pMMP‐2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.
Involvement of Src‐homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin‐like‐growth‐factor‐I‐receptor
Authors: GIORGETTI Sophie , PELICCI Pier Giuseppe , PELICCI Guilana , VAN OBBERGHEN Emmanuel 
Published: March 2005

Src homology/collagen (SHC) proteins are thought to participate in signaling through both receptor tyrosine kinases, such as the insulin receptor and the EGF (epidermal growth factor) receptor, and cytoplasmic tyrosine kinases, such as v‐src and v‐fps. Here we approached the insulin‐induced and the insulin‐like‐growth‐factor‐I‐induced (IGF‐I‐induced) phosphorylation of SHC proteins, and the possible role of these proteins in insulin and IGF‐I signaling. First, we showed that SHC proteins are phosphorylated on tyrosine residues upon insulin and IGF‐I treatment of fibroblasts transfected with a SHC cDNA construct. More important, ligand‐activated insulin and IGF‐I receptors phosphorylate SHC proteins in vitro, indicating that SHC proteins could be direct substrates for insulin and IGF‐I receptors. Further, insulin or IGF‐I treatment of SHC‐transfected fibroblasts leads to immunoprecipitation of SHC proteins with insulin‐receptor substrate 1 (IRS‐1).We next looked at the possible effect of SHC proteins on biological responses in SHC‐transfected fibroblasts. We found that the expression of exogenous SHC proteins results in an increased basal MEK (MAPK/ERK‐activating kinase) activity. Further, neither the basal nor the insulin‐induced or IGF‐I‐induced PtdIns‐3‐kinase activity were modified by expression of exogenous SHC proteins. These results illustrate that SHC proteins are implicated in the MAP(mitogen‐activated protein)‐kinase pathway, but not in that of PtdIns‐3‐kinase. Finally, we show that SHC‐transfected cells, unlike control cells, are able to advance into the early phases of the cell cycle, and are more sensitive to the growth‐promoting effect of insulin.In conclusion, SHC proteins are substrates for insulin and IGF‐I receptors, and would appear to function as early post‐receptor signaling components.
Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli
Authors: SEIPELT Irene , ENGELS Joachim W. 
Published: March 2005

A soluble domain of the interleukin(IL)‐2 receptor, the α chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine‐free IL‐2 receptor α chain (synIL‐2Rα) after BrCN cleavage and renaturation of the crude cleavage material, although the α chain is expressed as a deglycosylated, methionine‐free protein. The soluble receptor comprises amino acids 1–219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL‐2 and monoclonal antibodies as ligands.Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions.The deletion of a non‐bridging cysteine at position 192 in the synIL‐2Rα did not affect the renaturation yield of the receptor protein. Additionally a cysteine‐free and methionine‐free β‐galactosidase derivative was fused to the soluble synIL‐2Rα derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN‐cleavage material. It is suggested that cysteine impurities from cyanogen‐bromide‐cleaved peptides might interfere seriously with the refolding process of the synthetic IL‐2 receptor α‐subunit.
Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca2+‐ATPase by the non‐ionic detergent dodecylmaltoside
Authors: DE FORESTA Béatrice , HENAO Fernando , CHAMPEIL Philippe 
Published: March 2005

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca2+‐ATPase cycle by the non‐ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X‐100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209, 1023–1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20°C, showed that only one 45Ca2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45Ca2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [γ‐32P]ATP of the ATPase shifted towards 50‐fold higher Ca2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM‐perturbed ATPase requires the binding of this second, low‐affinity Ca2+. In order to assess this, samples of ATPase were intramolecularly cross‐linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediate with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613–4621]. Both in the absence and presence of DM, the cross‐linked enzyme occluded close to two Ca2+/phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high‐energy substrates, ATP or p‐nitrophenylphosphate (PNpP), were also found to shift towards higher Ca2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca2+/substrate hydrolyzed.As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca2+ binding. The loss of cooperativity of Ca2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca2+ binding.
Carbamoyl phosphate and ureagenesis are not involved in amino‐acid‐stimulated glycogenesis
Authors: GUSTAFSON Lori A. , ROMP Nancy , VAN WOERKOM George M. , MEIJER Alfred J. 
Published: March 2005

Amino acids are known to stimulate glycogen synthesis via an increase in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990)J. Biol. Chem. 265, 955–959]. It has recently been postulated, however, that carbamoyl phosphate, an intermediate of ureagenesis, can function as a substrate for glucose phosphorylation via carbamoyl‐phosphate:glucose phosphotransferase activity of the glucose‐6‐phosphatase system. This hypothesis would account for the stimulation of glycogenesis by amino acids such as glutamine and proline [Bode, A. M. & Nordlie, R. C. (1993)J. Biol. Chem. 268, 16298–16301].To further examine the role carbamoyl phosphate may play in glycogenesis, isolated hepatocytes were incubated under a variety of conditions to manipulate ureagenesis, glycogenesis and carbamoyl‐phosphate levels. Our data indicate that carbamoyl‐phosphate levels do not correlate with amino‐acid‐stimulated glycogenesis and that ureagenesis and glycogenesis are not competing metabolic pathways.
Phosphorylation of synthetic fragments of inhibitor‐2 of protein phosphatase‐1 by casein kinase‐1 and ‐2
Authors: MARIN Oriano , MEGGIO Flavio , SARNO Stefania , ANDRETTA Monica , PINNA Lorenzo A. 
Published: March 2005

The major phosphorylation site for both casein kinase‐2 (CK2) and casein kinase‐1 (CK1) in protein phosphatase‐1 (PP‐1) inhibitor‐2 (I‐2) is Ser86. Minor phosphorylation sites affected by either CK2 or CK1 are Ser120/Ser121 and Ser174, respectively. A synthetic peptide of 25 amino acids encompassing residues 67–93 of I‐2 is phosphorylated by either CK2 or CK1 at its seryl residue corresponding to Ser86 with higher Vmax and Km values similar to those of the intact protein (9 vs 7.2 μM and 14.2 vs 5.3 μM with CK2 and CK1, respectively). No detectable phosphorylation of this peptide which also includes the glycogen synthase kinase‐3 (GSK‐3) site (Thr72), could be observed with either GSK‐3 or p34cdc2 kinase whether or not its seryl residue equivalent to Ser86 had been previously phosphorylated by CK2. Shorter derivatives of I‐2(67–93), encompassing residues 72–93 and 78–93, are also readily phosphorylated by both CK1 and CK2, with phosphorylation efficiencies similar to those of the parent peptide.A synthetic heptadecapeptide reproducing the phosphoacceptor site around Ser120/Ser121 is phosphorylated by CK2, but not to any detectable extent by CK1, with a Km value fivefold higher than that of the corresponding pentadecapeptide including Ser86 (78–93).A synthetic pentadecapeptide (166–180) reproducing the phosphoacceptor site around Ser174 is phosphorylated by CK1 less efficiently than the pentadecapeptide including its main phosphorylation site (78–93) (Km 280 μM vs 33 μM). This peptide is readily phosphorylated by CK2 as well, although it lacks the canonical consensus sequence for CK2 and its Ser174 is almost unaffected by CK2 in intact I‐2.These data provide the clear‐cut demonstration that the consensus sequence with N‐terminal prephosphorylated residue(s), SerP/ThrP‐Xaa‐Xaa‐Ser/Thr, [Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W. & Roach, P. J. (1990) J. Biol. Chem. 265, 14264–14269; Meggio, F., Perich, J. W., Reynolds, E. C. & Pinna, L. A. (1991) FEBS Lett. 283, 303–306] is not always required to achieve efficient and high‐affinity phosphorylation by CK1. They also show that the specificity determinants for I‐2 phosphorylation by either CK2 or CK1, but not by GSK3, are entirely grounded on local structural features of the phosphoacceptor site, being only marginally affected by the overall structure of I‐2.