bluuebunny/crossref_metadata_2025 huggingface dataset:
Conversion of the Kunitz‐Type Module of Collagen VI into a Highly Active Trypsin Inhibitor by Site‐Directed Mutagenesis
Authors: Kohfeldt Eddie , Göhring Walter , Mayer Ulrike , Zweckstetter Markus , Holak Tad A. , Chu Mon‐Li , Timpl Rupert
Published: August 2004

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen α3(VI) chain was previously shown to lack inhibitory activity for proteases with trypsin‐like specificity and some other proteases. We have now prepared mutants in the binding loop region including the P1′ site (D2889 → A), the P2′ site (F2890 → R) and the P3 site (T2886 → P) and in a more remote region (W2907 → V) either as individual substitutions or combinations of them. These mutants were analyzed for their kinetics of binding to trypsin by surface plasmon resonance and for their capacity to inhibit various proteases. Single substitutions (D → A, T → P, W → V) showed an effect only for D → A which bound to trypsin with Kd= 0.25 μM. A 25–100‐fold increase in affinity was observed for the double mutants T → P/D → A and F → R/D → A and approached the affinity of aprotinin (Kd≈0.01 nM) in two different triple mutants. These affinities correlated well with the inhibitory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely identical improvement in inhibitory capacity was also observed for leucocyte elastase but not for thrombin. These data could be interpreted in terms of steric interferences or lack of hydrogen bonding of a few critical residues based on three‐dimensional structures available for the C5 domain.
Phosphorylation of Plant Proteins and the Identification of Protein‐Tyrosine Kinase Activity in Maize Seedlings
Authors: Trojanek Joanna , Ek Pia , Scoble Judith , Muszyńska Grażyna , Engström Lorentz
Published: August 2004

Phosphotyrosine was Found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins.

Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark‐grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40–65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).
The Evolutionary Conservation of a Novel Protein Modification, the Conversion of Cysteine to Serinesemialdehyde in Arylsulfatase from Volvox carteri
Authors: Selmer Thorsten , Hallmann Armin , Schmidt Bernhard , Sumper Manfred , Von Figura Kurt
Published: August 2004

A novel post‐translational protein modification has recently been described in two human sulfatases, by which a cysteine is replaced by a serinesemialdehyde (2‐amino‐3‐oxopropionic acid) residue [Schmidt, B., Selmer, T., Ingendoh, A. & von Figura, K. (1995) Cell 82, 271–278]. This cysteine is conserved among all known eukaryotic sulfatases. Here we report the presence of this modification in arylsulfatase from the green alga Volvox carteri. The evolutionary conservation of this novel protein modification between sulfatases of V. carteri and man lends further support to the assumption that this modification is required for the catalytic activity of sulfatases and may be present in all sulfatases of eukaryotic origin.
Nuclear‐Magnetic‐Resonance Determination of the Electron Self‐Exchange Rate Constant of Clostridium pasteurianum Rubredoxin
Authors: Gaillard Jacques , Zhuang‐Jackson Huayun , Moulis Jean‐Marc
Published: August 2004

The iron ion of rubredoxins efficiently exchanges one electron between the Fe(II) and Fe(III) oxidation states in mixtures of oxidized and reduced protein. The conditions under which the relaxation properties of the NMR signals can provide information about this exchange process have been worked out. The rate constant for the rubredoxin electron self‐exchange ranges between 1.5 × 105M−1 s−1 at 30°C and 3×105 M−1 s−1 at 30°C with an activation energy of the order of 24–30 kJ mol−1 in 50 mM potassium phosphate, pH 7. The increase of the electron self‐exchange rate constant with ionic strength suggests that neutralizing electrostatic repulsion between the active sites of two molecules further accelerates the already fast electron exchange.
The Antenna Complexes of the Purple Non‐Sulfur Photosynthetic Bacterium Rhodocyclus tenuis
Authors: Hu Qinghui , Brunisholz René A. , Frank Gerhard , Zuber Herbert
Published: August 2004

The photoreceptor complex (B885‐RC) and the peripheral antenna complex (B800–860) were isolated from photosynthetic membranes of the purple non‐sulfur bacterium Rhodocyclus tenuis DSM 109 using a detergent combination of Deriphate‐160 and octyl glucoside and subsequent linear sucrose gradient centrifugation. The two complexes were characterized by room‐temperature absorption, circular dichroism and fluorescence spectroscopy. The B800–860 complex has a more red‐shifted B860 absorbance band. The a β‐polypeptides were purified with a reverse‐phase HPLC system and resolved at a ratio of 1:1 in the B800–860 complex and at an overall ratio of 1:1 for the B885‐RC complex. The complete amino acid sequences of the α and β polypeptides of the B800–860 and B885‐RC complexes were determined by micro‐sequencing analysis and mass spectrometry. The B800–860–α polypeptide possesses an identical N‐terminal domain (the first 15 residues) to Rhodobacter sphaeroides B800–850–α polypeptide. The central hydrophobic and C‐terminal domains of the B800–860–α,β polypeptides show a number of B870/880‐like structural elements in which, of special interest, is the WWSEF cluster in the C‐terminal domain of the B800–860–α polypeptide which is very similar to the WWEF cluster in the same region of Rhodopseudomonas viridis B1015–α polypeptide. The more red‐shifted absorption characteristic of the 860‐nm bacteriochlorophylls could most probably be related to the B870/880‐like polypeptide features in the central hydrophobic domains and the C‐terminal domains of the B800–860–α,β polypeptides. The hydropathy plot of the B800–860–α,β polypeptide exhibits an extended C‐terminal hydrophobic segment indicative of a second membrane‐contacting domain, which has not been found in the antenna polypeptides of the purple bacteria with intracytoplasmic membranes. Further sequence analysis revealed the existence of multiple forms of the B885–α,β polypeptides, the B885–α1,α2 polypeptides, and the B885–β1,β2 polypeptides. The B885–α2 polypeptide shows an identical sequence to the B885–α1, polypeptide, but it is 12 amino acid residues shorter than the B885–α1, polypeptide at the C‐terminal. The two species of the B885–β1 polypeptides were identified as an identical sequence with only one amino acid residue variation at sequence position 34, where the B885–β1 has a valine residue and the B885–β2 polypeptide an isoleucine residue. The possible correlation between the intensity of the near‐infrared circular dichroic signal and the specific stuctural features of the α and β core antenna polypeptides is also discussed.
trans ‐Acting Factors, Detoxication Enzymes and Hepatitis B Virus Replication in a Novel Set of Human Hepatoma Cell Lines
Authors: Le Jossic Catherine , Glaise Denise , Corcos Laurent , Diot Christian , Dezier Jean‐François , Fautrel Alain , Guguen‐Guillouzo Christiane
Published: August 2004

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver‐specific mRNAs and DNA‐binding activity of ubiquitous and liver‐enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3‐methylcholanthrene‐inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA‐binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF‐1 and HNF‐4‐like DNA‐binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1‐like DNA‐binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer‐binding protein and HNF‐3‐like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis‐B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis‐B virus replication and virion production, in agreement with the notion that HNF‐1 activity is necessary for viral replication.

We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis‐B virus replication.
Cloning and Characterisation of Angiotensin‐Converting Enzyme from the Dipteran Species, Haematobia irritans exigua, and Its Expression in the Maturing Male Reproductive System
Authors: Wijffels Gene , Fitzgerald Clare , Gough Joanne , Riding George , Elvin Chris , Kemp David , Willadsen Peter
Published: August 2004

The angiotensin‐converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin‐affinity chromatography and ion‐exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41–42% amino acid identity to the mammalian testicular ACE. The active‐site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and Vmax values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin‐8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.
Purification of Escherichia coli Pro‐Haemolysin, and a Comparison with the Properties of Mature α‐haemolysin
Authors: Soloaga Ana , Ostolaza Helena , Goñi Félix M. , De La Cruz Fernando
Published: August 2004

Pro‐haemolysin (≈110 kDa), the inactive precursor of the membrane‐lytic toxin α‐haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro‐haemolysin forms aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro‐haemolysin can bind the lipid bilayers to a similar extent as α‐haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro‐haemolysin is totally unable to induce liposomal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of α‐haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca2+‐binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformation al change in the protein that involves the Ca2+‐binding domain.
NMR Investigation of the Solution Conformation of Oxidized Flavodoxin from Desulfovibrio vulgaris
Authors: Knauf Martin A. , Löhr Frank , Blümel Markus , Mayhew Stephen G. , Rüterjans Heinz
Published: August 2004

Desulfovibrio vulgaris flavodoxin has been investigated with a combination of homo‐ and hetero‐nuclear two‐dimensional and three‐dimensional NMR spectroscopy. The analysis of NOE, hydrogen exchange and J ‐coupling data led to a set of 1349 NOE, 63 hydrogen bond and 109 backbone φ‐angle restraints which were used to determine the solution structure of the oxidized flavodoxin applying the distance geometry program DIANA combined with restrained energy minimization methods. Flavodoxin in solution consists of a five‐stranded parallel β‐sheet which is pairwise flanked by four α‐helices. The solution structure has been compared with the known crystal structure. While the global fold is identical, differences have been detected concerning local conformations. In addition, protein‐bound water molecules have been localized by NOE effects which were detected in NMR experiments avoiding solvent suppression. The locations of these water molecules have been compared with those found in the X‐ray structure.
Amino Acid Sequence of Chicken Cu, Zn‐Containing Superoxide Dismutase and Identification of Glutathionyl Adducts at Exposed Cysteine Residues
Authors: Schininà M. Eugenia , Carlini Patrizia , Polticelli Fabio , Zappacosta Francesca , Bossa Francesco , Calabrese Lilia
Published: August 2004

The copper, zinc‐containing superoxide dismutase electromorphs from chicken erythrocytes have been isolated, their complete amino acid sequence determined and the identity of the protein moieties established. All electromorphs are constituted by a polypeptide chain made of 153 amino acid residues, corresponding to a molecular mass of 15598 Da. Accurate molecular mass determination by electrospray mass spectrometry of the separated electromorphs unequivocally proved that, in the chicken superoxide dismutase, either one or two cysteine residues/subunit are involved in a mixed disulfide with glutathione. The same post‐translational modification has been proven to occur in human superoxide dismutase. A different rate of S‐thiolation by endogenous glutathione was also demonstrated to be responsible for charge heterogeneity in cells. Effect of this modification on the catalytic and molecular properties of superoxide dismutases, and possible mechanisms for the S‐thiolation process, were also investigated and discussed.
Phosphorylated T Cell Receptor ζ‐chain and ZAP70 Tandem SH2 Domains form a 1:3 Complex in vitro
Authors: Weissenhorn Winfried , Eck Michael J. , Harrison Stephen C. , Wiley Don C.
Published: August 2004

The ζ polypeptide is part of the T cell antigen receptor (TCR). The ζ‐chain contributes to efficient cell‐surface expression of the TCR and accounts for part of its signal transduction capability. TCR recognition triggers a complex set of events that result in cellular activation. The protein tyrosine kinase (PTK) Lck phosphorylates the ζ‐chain, which in turn associates with another PTK, ZAP70, and stimulates its phosphorylation activity. Here we report the expression of the intracellular part of the ζ‐chain and its biochemical characterization. The recombinant protein does not dimerize by itself in solution. Circular‐dichroic analysis reveals a random coil conformation. ζ, phosphorylated using recombinant Lck, associates with recombinant ZAP70 tandem‐SH2 domains. All three T cell activation motifs in ζ bind ZAP70 tandem‐SH2 domains in vitro, forming a 1:3 complex. This result extends the picture, derived from earlier studies, of a mechanism for signal amplification
Mutation of the Conserved Cys165 Outside of the CuA Domain Destabilizes Nitrous Oxide Reductase but Maintains Its Catalytic Activity
Authors: Dreusch Andreas , Riester Joachim , Kroneck Peter M. H. , Zumft Walter G.
Published: August 2004

The single conserved Cys165 outside of the CuA domain of nitrous oxide reductase (N2OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, Cuz. The point mutation reduced the cellular level of N2OR 5–10‐fold compared to the level of the control strain. In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N2OR structural gene, nosZ, remained unaffected. The mutant enzyme was processed and exported into the periplasm like the wild‐type enzyme. Chemical analysis for sulfhydryl groups gave about nine ‐ SH groups/monomer of the apoenzyme prepared from the wild‐type enzyme, in accordance with the nine cysteine residues of the derived amino acid sequence. Eight ‐SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the enzyme Cys165 does not bind to Cuz, but may be part of a disulfide bridge essential for the stability of N2OR. Immediately downstream of the genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.
The Metabolism of Sphingo(glyco)lipids is Correlated with the Differentiation‐Dependent Autophagic Pathway in HT‐29 Cells
Authors: Ghidoni Riccardo , Houri Jean‐Jacques , Giuliani Attilia , Ogier‐Denis Eric , Parolari Elena , Botti Sara , Bauvy Chantal , Codogno Patrice
Published: August 2004

Recently it was demonstrated that the metabolism of both glycoproteins and sphingo(glyco)lipids is dependent upon the state of enterocytic differentiation of HT‐29 cells. Furthermore, it was shown that undifferentiated HT‐29 cells display an important autophagic sequestration, controlled by a heterotrimeric Gi3 protein. In order to correlate the metabolism of sphingo(glyco)lipids with the extent of autophagic sequestration, we have incubated undifferentiated and differentiated HT‐29 cells with tritium‐labelled GM1 ganglioside and sphingosine in the absence and presence of pertussis toxin (an inhibitor of autophagic sequestration) or asparagine (an inhibitor of autophagic vacuole maturation). In addition, undifferentiated HT‐29 cells transfected with a cDNA encoding the Gαi3 protein (cells expressing an amplified autophagic pathway) were labelled with both GM1 and sphingosine. The results show that the catabolism of sphingo(glyco)lipids is dramatically enhanced in parallel with the increase of the autophagic pathway while at the same time their biosynthesis is reduced. The inhibition of autophagy in both undifferentiated cells and αi3‐overexpressing cells restores sphingo(glyco)lipid metabolism, as normally expressed in differentiated cells, as well as in other mammalian cell types. We conclude that autophagy plays an important role in governing the metabolic fate of sphingo(glyco)lipids in HT‐29 cells. Since autophagy regulates the N‐linked glycoprotein metabolism in this cell line, our results corroborate the idea that glycolipid and glycoprotein metabolisms are controlled by similar mechanisms.
Developmental Expression of CYP2E1 in the Human Liver
Authors: Vieira Isabel , Sonnier Michelle , Cresteil Thierry
Published: August 2004

Cytochromes P‐450 are responsible for the biotransformation of drugs and other hydrophobic molecules by the liver. Several isoforms coexist which display an asynchronous onset during the perinatal period suggesting the involvement of multiple mechanisms of regulation. In this paper, we have shown that the CYP2E1 protein and its associated activity could not be detected in the fetal liver and rise during the first few hours following birth independently of the gestational age (between 25–40 weeks). During this period, the CYP2E1 RNA content remains fairly low: the stabilization of the low amount of existing CYP2E1 protein by endogenous ketone bodies could explain the early neonatal rise of the protein level. From 1 month to 1 year, the protein content gradually increases and is accompanied by the accumulation of CYP2E1 RNA, suggesting a transcriptional activation of the gene during the late neonatal period.

We examined the methylation status of CpG residues in the 5′ flanking region, first exon and first intron of CYP2E1 gene cleaved with Hpa II/Msp I. Genomic DNA from fetal liver shows several hypermethylated spots in the first‐exon–first‐intron region, which progressively disappear in neonatal samples. We conclude that during the neonatal period, the accumulation of hepatic CYP2E1 RNA is correlated with the degree of methylation at the 5′ end of the CYP2E1 gene.
Functional Characteristics of a Variant Thyrotropin Receptor
Authors: Tonacchera Massimo , Cetani Filomena , Costagliola Sabine , Van Sande Jacqueline , Refetoff Samuel , Vassart Gilbert
Published: August 2004

A claim has been made that a variant of the human thyrotropin receptor in which Pro52 is replaced by Thr([Thr52]thyrotropinreceptor) is associated with autoimmune thyroid diseases and displays increased responsiveness to thyrotropin. We have analysed the functional characteristics of this variant receptor. Equivalent numbers of of the wild type and of the variant thyrotropin receptor, measured both by 125I‐thyrotropin binding and by flow cytofluorimetry, were transiently expressed in COS‐7 cells. Under these conditions, the two receptors showed the same degree of constitutive activity for the cAMP pathway, the same affinity for bovine thyrotropin, and a virtually identical responsiveness to bovine thyrotropin for activation of both the cAMP and inositol‐phosphate regulatory pathways. Our results show that the [Thr52]thyrotropin receptor variant of the human thyrotropin receptor, which is present in the 12% of the population, does not affect receptor function and represents most likely a simple polymorphism.
Dissociation of the Complex between the Neuroendocrine Chaperone 7B2 and Prohormone Convertase PC2 is not Associated with proPC2 Maturation
Authors: Braks Joanna A. M. , Van Horssen A. Martin , Martens Gerard J. M.
Published: August 2004

7B2 is a highly conserved neuroendocrine protein that is associated with the proform of the prohormone convertase PC2 in the early stages of the secretory pathway in intermediate pituitary cells of Xenopus laevis. Subsequent processing of 7B2 and dissociation of the 7B2/proPC2 complex is thought to be associated with the conversion of proPC2 to the mature enzyme. Here, we report that, in both Xenopus and mouse intermediate pituitary cells, proPC2 maturation does not take place when the proenzyme is associated with the 7B2 precursor and that, in contrast to the previous notion, dissociation of the complex between proPC2 and the N‐terminal 7B2 fragment precedes, and is thus not directly linked to, proPC2 maturation. In vitro, conversion of newly synthesized proPC2 was efficiently blocked by recombinant 7B2 and studies with truncation mutants indicated that a short segment in the C‐terminal region of 7B2 is necessary and sufficient for this inhibitory effect. Our results indicate that, after 7B2 precursor processing and dissociation of the N‐terminal fragment, the C‐terminal fragment of 7B2 may remain associated with proPC2, thereby preventing autocatalytic conversion of the proenzyme until the appropriate site for activation in the secretory pathway is reached.
Impairment of DNA Replication within One Cell Cycle after Seeding of Cells in the Presence of a Polyamine‐Biosynthesis Inhibitor
Authors: Fredlund Jan O. , Oredsson Stina M.
Published: August 2004

Chinese hamster ovary (CHO) cells in the plateau phase were seeded in the absence or presence of 5 mM 2‐difluoromethylornithine (F2MeOrn), an enzyme‐activated irreversible inhibitor of ornithine decarboxylase. The thymidine analogue bromodeoxyuridine (BrdUrd, 5 μM) was added to the culture medium 30 min before sampling of the cells, which occurred 1–17 h after seeding. Using flow cytometry, coupled with an indirect immunofluorescence technique, which utilized monoclonal BrdUrd and secondary fluorescein‐isothiocyanate‐conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. To determine if there was a perturbation in the progression of cells through the S phase, the distribution of BrdUrd‐labelled cells in the S phase was evaluated in two ways: (a) by calculating the mean DNA content of BrdUrd‐labelled cells in relation to the mean DNA contents of G1 and G2 cells (relative movementzero) and (b) by studying DNA histograms of BrdUrd‐labelled cells. By using both evaluation methods, we show that DNA replication was impaired during the first cell cycle that was initiated after seeding CHO cells in the presence of F2MeOrn. The cells appeared to enter the S phase normally but were then delayed in their progression through this phase. The impairment of F2MeOrn treatment on DNA replication was apparent at 9 h after seeding, a time point at which the putrescine pool was depleted, the spermidine pool was approximately halved, and the spermine pool was unaffected, when compared to corresponding pools of control cells. When cells were seeded in the presence of F2MeOrn and putrescine, the effect on DNA replication was prevented. The rates of incorporation of [3H]uridine and [3H]leucine into RNA and protein, respectively, were the same in control and in F2MeOrn‐treated cells for at least up to 11 h after seeding.
The effect of Linoleic Acid on pH Inside Sodium bis(2–ethylhexyl)sulfosuccinate Reverse Micelles in Isooctane and on the Enzymic Activity of Soybean Lipoxygenase
Authors: Rodakiewicz‐Nowak Janina , Maślakiewicz Pawel , Haber Jerzy
Published: August 2004

The effective pH of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) reverse micelles (pHrm), containing buffers of different pH (pHst) and various amounts of linoleic acid, was studied within the range of compositions used to study the activity of soybean lipoxygenase in reverse micelles. Significant shifts of pHrm versus pHst were observed for the solutions of relatively higher pHst, dependent on linoleic acid and buffer concentrations. The effect diminished as pHst became closer to 7. When low‐ionic‐strength buffers were added to AOT solutions in isooctane, a significant buffering effect of linoleic acid in reverse micelles was observed. Solubilization of > 3 mM linoleic acid in micellar solutions containing 25 mM buffers gave the observed pHrm values almost independent of pHst. This effect diminished with the ionic strength of the buffering solution, but did not vanish even at 200 mM buffer. The observed effects result from the balance between ionization of linoleic acid and its partition between the water pool and the micellar interface.

The enzymic activity of soybean lipoxygenase in the AOT reverse micellar solutions of the determined pHrm values was also studied. A significant reduction of the kinetics of the enzymic activity was observed, for all studied reverse micellar solutions. Changes of pHrm, caused by the presence of acidic substrate (linoleic acid) do not explain the observed reduction of activity directly through the effect on the enzyme. Due to unfavourable partition of the substrate between the microphases present in the systems, enhanced by reduction of pH at higher total concentrations of linoleic acid, the saturation of the enzyme with the substrate was not observed in the system and is difficult to attain experimentally in reverse micelles.

A shift of the lipoxygenase activity/pHrm profile but negligible shift of the activity/pHst profile, with respect to aqueous buffer solutions, were observed. This indicates that either the information given by pH indicators used does not reveal the true pH of the enzyme in these reverse micelles, or that the sample of the enzyme is pH‐insensitive over a broader pH range than results from the relationship observed for linoleic acid in aqueous solutions. The latter conclusion is consistent with the data on lipoxygenase activity towards linoleyl sulfate in aqueous solutions [Bild, G. S., Ramadoss, C. S. & Axelrod, B. (1977) Lipids 12, 732–735]. Conditions for measuring pH‐independent lipoxygenase activity in reverse micelles are discussed.
Translocation and Aggregation of Hepatic Glycogen Synthase During the Fasted‐to‐Refed Transition in Rats
Authors: Fernández‐Novell Josep M. , Roca Angela , Bellido David , Vilaró Senen , Guinovart Joan J.
Published: August 2004

Changes in the activation state and intracellular distribution of liver glycogen synthase have been studied during the fasted‐to‐refed transition in rats. Glycogen synthase activity and activation state were measured in supernatants and pellets obtained after centrifugation of liver homogenates at 9200g. Upon refeeding, the glycogen synthase activity ratio increased, in a time‐dependent manner, in both fractions. The total activity of the enzyme decreased in supernatants and was quantitatively recovered in the pellets. Therefore, refeeding induced both the activation of glycogen synthase and its translocation from the soluble to the pelletable fraction. Immunocytochemical evidence indicates that refeeding induced the formation of clusters of glycogen synthase, which were recovered in the 9200g sediments. However, the enzyme clusters did not locate with the glycogen particles in the pelletable fraction. The glycogen synthase activation state responded almost as an on‐off switch to changes in the intracellular glucose 6‐phosphate concentration in the range 0.2–0.3 mM. The amount of enzyme present in the pellets correlated linearly with the intracellular glucose 6‐phosphate levels. These results indicate that glucose 6‐phosphate is the key signal for both the activation and changes in intracellular localization of hepatic glycogen synthase in vivo.
Cloning of the maoA Gene that Encodes Aromatic Amine Oxidase of Escherichia coli W3350 and Characterization of the Overexpressed Enzyme
Authors: Steinebach Vincent , Benen Jac A. E. , Bader Rechien , Postma Pieter W. , De Vries Simon , Duine Johannis A.
Published: August 2004

The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes [Sugino, H., Sasaki, M., Azakami, H., Yamashita, M. & Murooka, Y. (1992) J. Bacteriol. 174, 2485–2492]. The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E. coli TG2.

The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)‐containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines. However, high‐resolution anion‐exchange chromatography revealed that the preparation was heterogeneous. The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474m/A340nm ratios and TPQ/subunit ratios. Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N‐terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ. Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms.
A TGACG Motif Mediates Growth‐Hormone‐Factor‐1/Pituitary‐Transcriptional‐Activator‐1‐Dependent cAMP Regulation of the Rainbow Trout Growth‐Hormone Promoter
Authors: Argenton Francesco , Bernardini Serena , Puttini Stefania , Colombo Lorenzo , Bortolussi Marino
Published: August 2004

The mechanisms involved in the regulation of the rainbow trout growth hormone (tGH) gene promoter by the pituitary‐specific transcription factor GHF1 (growth hormone factor 1), also called Pit1 (pituitary transcriptional activator 1), and cAMP have been investigated in mammalian and fish cells. The ‐340 to +24 5′‐flanking region of the tGH gene fused to the luciferase gene was activated in rat pituitary GC cells and in HeLa cells cotransfected with an effector plasmid encoding rat GHF1. GC cell nuclear extracts produced four GHF1‐specific footprints (sites F1 to F4) on the tGH promoter, each containing multiple W4NCAT (W, A or T) or closely related motifs. Mutational analysis performed in GC cells indicated that the proximal F1 site alone can direct transcription, but that the region encompassing the F2 and F3 sites is necessary for optimal activation and contains a TGACG motif (cAMP‐response element, CRE) confering cAMP responsiveness. The role of the TGACG motif in mediating cAMP regulation of the tGH promoter was confirmed in primary cultures of trout pituitary cells. Cotransfection studies in carp EPC cells using an effector plasmid encoding trout GHF1 demonstrated the GHF1 dependence of cAMP stimulation. Gel shift and southwestern experiments revealed nuclear proteins of 43 kDa and 30 kDa in GC and fish cells, respectively, that bind specifically to the tGH CRE, suggesting the involvement of CRE‐binding‐protein/activating‐transcription‐factor‐1‐related peptides in cAMP response. Incidentally, and in contrast with previous reports, we found the rat GH promoter, that lacks TGACG motifs, unresponsive to cAMP. Thus, the cAMP stimulation of the tGH gene is more similar to its human counterpart, that is also GHF1 dependent and mediated by TGACG motifs in the promoter. It is suggested that control of GH gene expression has evolved modularly, through various assortments of the same regulatory units, rather than molecularly, through innovative units.
Flavin Motion in p‐Hydroxybenzoate Hydroxylase
Authors: Van Der Bolt Frank J. T. , Vervoort Jacques , Van Berkel Willem J. H.
Published: August 2004

The side chain of Tyr222 in p ‐hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222→Phe mutant, [F222]p ‐hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4‐hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222→Ala mutant, [A222]p ‐hydroxybenzoate hydroxylase, was investigated.

Replacement of Tyr→222 by Ala impairs the binding of the physiological substrate 4‐hydroxybenzoate and the substrate analog 4‐aminobenzoate. With these compounds, [A222]p ‐hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p ‐hydroxybenzoate hydroxylase tightly interacts with 2,4‐dihydroxybenzoate and 2‐hydroxy‐4‐aminobenzoate. Crystallographic data [Schreuder, H. A., Mattevi, A., Oblomova, G., Kalk, K. H., Hoi, W. G. J., van der Bolt, F. J. T. & van Berkel, W. J. H (1994) Biochemistry 33, 10161–10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen‐bond interaction between the 2‐hydroxy group of the substrate analogs and N3 of the flavin. [A222]p ‐Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4‐trihydroxybenzoate from 2,4‐dihy‐droxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222→Ala mutant shifts the equilibrium of flavin conformers towards the productive ‘in’ position.

[A222]p ‐Hydroxybenzoate hydroxylase converts 2‐fluoro‐4‐hydroxybenzoate to 2‐fluoro‐3,4‐dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p ‐hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild‐type. Spectral studies suggest that wild‐type and [A222]p ‐hydroxybenzoate hydroxylase bind 2‐fluoro‐4‐hydroxybenzoate in the phenolate form with the flavin ring preferring the ‘out’ conformation. Despite activation of the fluorinated substrate and in contrast to the wild‐type enzyme, [A222]p ‐hydroxybenzoate hydroxylase largely produces hydrogen peroxide.

The effector specificity of p ‐hydroxybenzoate hydroxylase is not changed by the Tyr222→‐Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein‐substrate interactions at the re ‐side of the flavin ring.
Formation of a Novel Enzymatic Metabolite of Leukotriene A4 in Tissues of Xenopus laevis
Authors: Strömberg Filippa , Hamberg Mats , Rosenqvist Urban , Dahlén Sven‐Erik , Haeggström Jesper Z.
Published: August 2004

Leukotriene‐A4 hydrolase catalyzes the final step in the biosynthesis of the potent proinflammatory mediator leukotriene B4. Previously, leukotriene‐A4 hydrolase has been characterized from human, mouse and rat sources, i.e. only from mammalian species. In the present investigation, expression of leukotriene‐A4 hydrolase was studied in organs of Xenopus laevis. Enzyme activity was found in all nine organs tested with the highest levels in the intestine and the reproductive organs, i.e. oocytes and testes, previously unrecognized rich sources of the enzyme. No immunoreactive leukotriene‐A4 hydrolase was detected in Western blots of 10 000×g supernatants of X. laevis organ homogenates, using a polyclonal antiserum raised against human leukotriene‐A4 hydrolase. Likewise, Northern blot analysis of liver total RNA did not detect Xenopus leukotriene‐A4 hydrolase mRNA using a human cDNA probe. These results indicate significant structural differences between the human and toad enzymes.

Incubations of 10 000×g supernatants of organ homogenates with leukotriene A4 revealed the formation of a novel metabolite, denoted compound X. Conversion of leukotriene A4 into compound X was due to an enzymatic activity as judged by its protein dependence, heat sensitivity, and resistance to ultrafiltration, and this activity appeared to be linked, directly or indirectly, to leukotriene‐A4 hydrolase.

From data obtained by ultraviolet spectrophotometry, gas chromatography coupled to mass spectrometry, ultraviolet‐induced isomerization, and comparison with a synthetic standard, compound X was assigned the structure 5S,12R‐dihydroxy‐6,10‐trans 8,14‐cis‐eicosatetraenoic acid. Finally, compound X was found to exhibit contractile activity in guinea‐pig lung parenchyma, apparently elicited via a leukotriene B receptor.
Non‐Structural Protein 3 of Hepatitis C Virus Inhibits Phosphorylation Mediated by cAMP‐Dependent Protein Kinase
Authors: Borowski Peter , Heiland Max , Oehlmann Katrin , Becker Bettina , Kornetzky Lutz , Feucht Hubert , Laufs Rainer
Published: August 2004

Inspection of the amino acid sequence of the non‐structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487‐Arg‐Gly‐Arg‐Thr‐Gly‐Arg‐Gly‐Arg‐Arg‐Gly‐Ile‐Tyr‐Arg1500, located in the non‐structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat‐stable inhibitor of cAMP‐dependent protein kinase (PKA) and to the autophos‐phorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5‐kDa fragment of NS3 that consists of residues 1189–1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV‐polyprotein‐(1189–1525) influences PKA in a mixed‐inhibition‐type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV‐polyprotein‐(1189–1525). Binding studies with PKA and the non‐hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV‐derived peptides or HCV‐polyprotein‐(1189–1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.
Conformational Transitions within the Calmodulin‐Binding Site of Bordetella pertussis Adenylate Cyclase Studied by Time‐Resolved Fluorescence of Trp242 and Circular Dichroism
Authors: Bouhss Ahmed , Vincent Michel , Munier Hélène , Gilles Anne‐Marie , Takahashi Masayuchi , Bârzu Octavian , Danchin Antoine , Gallay Jacques
Published: August 2004

The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin‐binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by time‐resolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin.
Similarities in the Architecture of the Active Sites of Ni‐Hydrogenases and Fe‐Hydrogenases Detected by Means of Infrared Spectroscopy
Authors: Van Der Spek Trienke M. , Arendsen Alexander F. , Happe Randolph P. , Yun Suyong , Bagley Kimberly A. , Stufkens Derk J. , Hagen Wilfred R. , Albracht Simon P. J.
Published: August 2004

Three groups that absorb in the 2100–1800‐cm−1 infrared spectral region have recently been detected in Ni‐hydrogenase from Chromatium vinosum [Bagley, K. A., Duin, E. C., Roseboom, W., Albracht, S. P. J. & Woodruff, W. H. (1995) Biochemistry 34, 5527–5535]. To assess the significance and generality of this observation, we have carried out an infrared‐spectroscopic study of eight hydrogenases of three different types (nickel, iron and metal‐free) and of 11 other iron‐sulfur and/or nickel proteins. Infrared bands in the 2100–1800‐cm−1 spectral region were found in spectra of all Ni‐hydrogenases and Fe‐hydrogenases and were absent from spectra of any of the other proteins, including a metal‐free hydrogenase. The positions of these bands are dependent on the redox state of the hydrogenase. The three groups in Ni‐hydrogenases that are detected by infrared spectroscopy are assigned to the three unidentified small non‐protein ligands that coordinate iron in the dinuclear Ni/Fe active site as observed in the X‐ray structure of the enzyme from Desulfovibrio gigas [Volbeda, A., Charon, M.‐H., Piras, C., Hatchikian, E. C., Frey, M. & Fontecilla‐Camps, J. C. (1995) Nature 373, 580–587]. It is concluded that these groups occur exclusively in metal‐containing H2‐activating enzymes. It is proposed that the active sites of Ni‐hydrogenases and of Fe‐hydrogenases have a similar architecture, that is required for the activation of molecular hydrogen.
Molecular Cloning and Characterization of a Transmembrane Surface Antigen in Human Cells
Authors: Li Erqiu , Bestagno Marco , Burrone Oscar
Published: August 2004

The mouse mAb 6C6, raised against a plasma‐membrane preparation from human breast‐cancer cells, reacts with an antigen that appears to be overexpressed in human breast cancers and other human tumors. Here we describe the cDNA cloning and characterization of the antigen recognized by the 6C6 mAb. The isolated cDNA clone encodes a protein of 246 amino acids, with a predicted molecular mass of 27991 Da. The protein contains three amino‐terminal hydrophobic regions, which could represent transmembrane domains, and a hydrophilic carboxy‐terminal region, which we show to be extracellular. The identity of the protein encoded by the cloned cDNA as the 6C6 antigen was confirmed by in vitro translation and immunoprecipitation experiments, and by transfection into cell lines that do not react with the 6C6 mAb, which resulted in the expression of a 28‐kDa surface protein that was recognized by the antibody. The 6C6 antigen appears to be a type II transmembrane protein, with multiple membrane‐spanning domains and a long extracellular non‐glycosylated carboxy‐terminal domain, to which the 6C6 epitope has been mapped. The overall structure of the protein and weak amino acid similarities with a family of multiple‐transmembrane‐spanning‐domain proteins that includes some antigens (such as L6, CD63/ME491 and CO‐029) that are overexpressed in tumor cells, suggest that the 6C6 antigen may belong to this family of proteins.
Acceptor Substrate Specificity of a Cloned GD3 Synthase that Catalyzes the Biosynthesis of Both GD3 and GD1c/GT1a/GQ1b
Authors: Nara Kiyomitsu , Watanabe Yumiko , Kawashima Ikuo , Tai Tadashi , Nagai Yoshitaka , Sanai Yutaka
Published: August 2004

To address the role of α2,8‐sialyltransferase (GD3 synthase) in the biosynthesis of gangliosides, we examined the substrate specificity of the enzyme. In the ganglioside synthesis pathway, it has been generally accepted that sialyltransferase II (SAT II) catalyzes the production of GD3 from GM3, and sialyltransferase V (SAT V) catalyzes the production of GD1c/GT1a/GQ1b from GM1b/GD1a/GT1b. However, acceptor specificity of the cloned GD3 synthase that was isolated from human melanoma cells [Nara, K., Watanabe, Y., Maruyama, K., Kasahara, K., Nagai, Y. & Sanai, Y. (1994) Proc. Natl Acad. Sci. USA 91, 7952–7956] has revealed that this enzyme utilized the gangliosides containing the terminal Sia(α2–3)Gal structure of the carbohydrate moiety, which includes GM3, GM1b, GD1a, and GT1b as exogenous substrates. Kinetic data also showed that the enzyme was able to utilize both GM3 and GM1b/GD1a/GT1b as acceptor substrates. These data indicate that the enzyme catalyzes the formation of not only GD3 but also GD1c, GT1a, and GQ1bin vitro. Furthermore, by transfection of the cloned human α2,8‐sialyltransferase cDNA, transient and stable expression of GT1 a and GQ1b was also observed in COS‐7 cells and Swiss 3T3 cells that originally lacked SAT II and SAT V activities. These observations indicate that the enzyme has both SAT II and SAT V activities in vivo.